WO2023071934A1 - 新型的crispr酶和系统以及应用 - Google Patents

新型的crispr酶和系统以及应用 Download PDF

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WO2023071934A1
WO2023071934A1 PCT/CN2022/126671 CN2022126671W WO2023071934A1 WO 2023071934 A1 WO2023071934 A1 WO 2023071934A1 CN 2022126671 W CN2022126671 W CN 2022126671W WO 2023071934 A1 WO2023071934 A1 WO 2023071934A1
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nucleic acid
sequence
cas
target
protein
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PCT/CN2022/126671
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French (fr)
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李珊珊
孙洁
赵庆芝
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山东舜丰生物科技有限公司
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C12N9/22—Ribonucleases RNAses, DNAses
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C—CHEMISTRY; METALLURGY
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/20—Type of nucleic acid involving clustered regularly interspaced short palindromic repeats [CRISPRs]

Definitions

  • the invention relates to the field of gene editing, in particular to the technical field of clustered regularly interspaced short palindromic repeats (CRISPR).
  • CRISPR clustered regularly interspaced short palindromic repeats
  • the present invention relates to a novel CRISPR enzyme (or, referred to as CRISPR protein, Cas effector protein, Cas enzyme or Cas protein), fusion proteins comprising such proteins, and nucleic acid molecules encoding them.
  • the present invention also relates to complexes and compositions for nucleic acid editing (eg, gene or genome editing), which comprise the Cas protein or fusion protein of the present invention, or nucleic acid molecules encoding them.
  • CRISPR/Cas technology is a widely used gene editing technology. It uses RNA guidance to specifically bind to target sequences on the genome and cuts DNA to generate double-strand breaks. It uses biological non-homologous end joining or homologous recombination for site-directed gene editing.
  • the CRISPR/Cas9 system is the most commonly used type II CRISPR system, which recognizes the PAM motif of 3’-NGG and performs blunt-end cleavage of the target sequence.
  • the CRISPR/Cas Type V system is a newly discovered CRISPR system, which has a 5'-TTN motif and performs cohesive end cleavage on target sequences, such as Cpf1, C2c1, CasX, and CasY.
  • target sequences such as Cpf1, C2c1, CasX, and CasY.
  • the different CRISPR/Cas that currently exist have different advantages and disadvantages.
  • Cas9, C2c1, and CasX all require two RNAs as guide RNAs
  • Cpf1 requires only one guide RNA and can be used for multiple gene editing.
  • CasX has a size of 980 amino acids, while common Cas9, C2c1, CasY and Cpf1 are usually around 1300 amino acids in size.
  • PAM sequences of Cas9, Cpf1, CasX, and CasY are complex and diverse, while C2c1 recognizes a strict 5'-TTN, so its target site is easier to predict than other systems, thereby reducing potential off-target effects.
  • the inventor of the present application unexpectedly discovered a novel endonuclease (Cas enzyme) after a large number of experiments and repeated explorations. Based on this discovery, the present inventors developed a new CRISPR/Cas system and a gene editing method and a nucleic acid detection method based on the system.
  • the present invention provides a kind of Cas protein
  • described Cas protein is the effector protein in CRISPR/Cas system, and in the present invention, it is called Cas-sf0005 (amino acid sequence is as shown in SEQ ID No.1) , Cas-sf9417 (amino acid sequence as shown in SEQ ID No.4), Cas-sf8553 (amino acid sequence as shown in SEQ ID No.7) and Cas-sf1846 (amino acid sequence as shown in SEQ ID No.10), evolution Tree analysis showed that the above Cas proteins belonged to similar proteins.
  • the Cas protein amino acid sequence has at least 80%, at least 85%, at least 90% compared with SEQ ID No.1 or SEQ ID No.4 or SEQ ID No.7 or SEQ ID No.10 %, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity, and substantially retains its origin from The biological function of the sequence.
  • the Cas protein amino acid sequence has one or more amino acid substitutions and deletions compared with SEQ ID No.1 or SEQ ID No.4 or SEQ ID No.7 or SEQ ID No.10 or added sequence; and substantially retains the biological function of the sequence it is derived from; the substitution, deletion or addition of one or more amino acids includes 1, 2, 3, 4, 5, 6 , 7, 8, 9 or 10 amino acid substitutions, deletions or additions.
  • the structure of a protein can be changed without adversely affecting its activity and functionality, for example, one or more conservative amino acid substitutions can be introduced in the amino acid sequence of the protein without affecting the activity and/or functionality of the protein molecule Or three-dimensional structure adversely affected.
  • conservative amino acid substitutions are clear to those skilled in the art.
  • the amino acid residue can be replaced with another amino acid residue belonging to the same group as the site to be substituted, that is, a non-polar amino acid residue is used to replace another non-polar amino acid residue, and a non-polar amino acid residue is replaced with a polar uncharged amino acid residue.
  • amino acid residues for another polar uncharged amino acid residue substituting a basic amino acid residue for another basic amino acid residue, and substituting an acidic amino acid residue for another acidic amino acid residue.
  • Such substituted amino acid residues may or may not be encoded by the genetic code.
  • Conservative substitutions in which one amino acid is replaced by another amino acid belonging to the same group fall within the scope of the present invention as long as the substitution does not result in the inactivation of the biological activity of the protein. Therefore, the protein of the present invention may contain one or more conservative substitutions in the amino acid sequence, and these conservative substitutions are preferably produced according to Table 1.
  • the present invention also encompasses proteins that also contain one or more other non-conservative substitutions, as long as the non-conservative substitutions do not significantly affect the desired function and biological activity of the protein of the present invention.
  • Conservative amino acid substitutions can be made at one or more predicted non-essential amino acid residues.
  • a “nonessential” amino acid residue is one that can be altered (deleted, substituted or substituted) without altering biological activity, whereas an "essential” amino acid residue is required for biological activity.
  • a “conservative amino acid substitution” is one in which an amino acid residue is replaced by an amino acid residue with a similar side chain. Amino acid substitutions can be made in the non-conserved regions of the aforementioned Cas proteins. Generally, such substitutions are not made to conserved amino acid residues, or to amino acid residues located within conserved motifs, where such residues are required for protein activity. However, those skilled in the art will appreciate that functional variants may have fewer conservative or non-conservative changes in conserved regions.
  • proteins that have one or more amino acid residues altered from the N and/or C terminus of the Cas protein while retaining its desired functional activity are also within the scope of the present invention.
  • These changes may include those introduced by modern molecular methods such as PCR, which involves PCR amplification that alters or extends protein coding sequences by virtue of the inclusion of amino acid coding sequences in the oligonucleotides used in PCR amplification.
  • proteins may be altered in various ways, including amino acid substitutions, deletions, truncations and insertions, and methods for such manipulations are generally known in the art.
  • amino acid sequence variants of the above proteins can be prepared by mutating the DNA. It can also be accomplished by other forms of mutagenesis and/or by directed evolution, for example, using known mutagenesis, recombination and/or shuffling methods, in combination with relevant screening methods, for single or multiple amino acid substitutions, deletions and/or insertions.
  • these minor amino acid changes in the Cas protein of the present invention can occur (such as naturally occurring mutations) or be produced (such as using r-DNA technology) without loss of protein function or activity. If these mutations occur in the catalytic domain, active site, or other functional domains of the protein, the properties of the polypeptide may change, but the polypeptide may retain its activity. Smaller effects can be expected if the mutations present are not close to the catalytic domain, active site, or other functional domains.
  • Those skilled in the art can identify the essential amino acids of the Cas protein of the present invention according to methods known in the art, such as site-directed mutagenesis or protein evolution or bioinformatics analysis.
  • the catalytic domain, active site, or other functional domains of a protein can also be determined by physical analysis of structure, such as by techniques such as nuclear magnetic resonance, crystallography, electron diffraction, or photoaffinity labeling, binding to putative key sites. Point amino acid mutations are identified.
  • the Cas protein contains the amino acid sequence shown in SEQ ID No.1 or SEQ ID No.4 or SEQ ID No.7 or SEQ ID No.10.
  • the Cas protein is the amino acid sequence shown in SEQ ID No.1 or SEQ ID No.4 or SEQ ID No.7 or SEQ ID No.10.
  • the Cas protein is derivatized with the same biological function as the protein having the sequence shown in SEQ ID No.1 or SEQ ID No.4 or SEQ ID No.7 or SEQ ID No.10 protein.
  • the biological functions include, but are not limited to, the activity of binding to guide RNA, endonuclease activity, and the activity of binding and cutting to a specific site of the target sequence under the guidance of guide RNA, including but not limited to Cis cleavage activity and Trans cleavage active.
  • the present invention also provides a fusion protein, which includes the above-mentioned Cas protein and other modified parts.
  • the modification moiety is selected from another protein or polypeptide, a detectable label, or any combination thereof.
  • the modification moiety is selected from an epitope tag, a reporter gene sequence, a nuclear localization signal (NLS) sequence, a targeting moiety, a transcriptional activation domain (e.g., VP64), a transcriptional repression domain (e.g., KRAB domain or SID domain), a nuclease domain (e.g., Fok1), and a domain having an activity selected from the group consisting of: nucleotide deaminase, methylase activity, demethylase, transcription activation activity, transcription repression activity, transcription release factor activity, histone modification activity, nuclease activity, single-stranded RNA cleavage activity, double-stranded RNA cleavage activity, single-stranded DNA cleavage activity, double-stranded DNA cleavage activity, and nucleic acid binding activity; and any combination thereof.
  • the NLS sequence is well known to those skilled in the art, examples of which include, but are not limited to,
  • the NLS sequence is located at, near or near the end of the Cas protein of the present invention (for example, the N-terminus, the C-terminus or both ends).
  • epitope tag is well known to those skilled in the art, including but not limited to His, V5, FLAG, HA, Myc, VSV-G, Trx, etc., and those skilled in the art can choose other suitable tags Bit labeling (eg, purification, detection or tracking).
  • the reporter gene sequence is well known to those skilled in the art, examples of which include but are not limited to GST, HRP, CAT, GFP, HcRed, DsRed, CFP, YFP, BFP and the like.
  • the fusion protein of the present invention comprises a domain capable of binding to a DNA molecule or an intracellular molecule, such as maltose binding protein (MBP), the DNA binding domain (DBD) of Lex A, the DBD of GAL4, etc.
  • MBP maltose binding protein
  • DBD DNA binding domain
  • GAL4 GAL4
  • the fusion protein of the invention comprises a detectable label, such as a fluorescent dye, such as FITC or DAPI.
  • the Cas protein of the present invention is optionally coupled, conjugated or fused to the modification moiety through a linker.
  • the modification part is directly connected to the N-terminal or C-terminal of the Cas protein of the present invention.
  • the modified part is connected to the N-terminal or C-terminal of the Cas protein of the present invention through a linker.
  • linkers are well known in the art, examples of which include, but are not limited to, include one or more (e.g., 1, 2, 3, 4 or 5) amino acids (e.g., Glu or Ser) or amino acid derivatives (eg, Ahx, ⁇ -Ala, G ABA or Ava), or PEG, etc.
  • the Cas protein, protein derivative or fusion protein of the present invention is not limited by its production method, for example, it can be produced by genetic engineering method (recombinant technology), and can also be produced by chemical synthesis method.
  • the invention provides an isolated polynucleotide comprising:
  • nucleotide sequence described in any one of (a)-(e) is codon optimized for expression in prokaryotic cells. In one embodiment, the nucleotide sequence described in any one of (a)-(e) is codon optimized for expression in eukaryotic cells.
  • said polynucleotide is preferably single-stranded or double-stranded.
  • the present invention provides an engineered direct repeat sequence that forms a complex with the aforementioned Cas protein.
  • the direct repeat sequence is connected with the guide sequence capable of hybridizing with the target sequence to form a guide RNA (guide RNA or gRNA).
  • the hybridization of the target sequence to the gRNA represents at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96% of the nucleic acid sequence of the target sequence and the gRNA , 97%, 98%, 99%, or 100% identity, so that they can hybridize to form a complex; or there are at least 12, 15, 16, 17, 18 nucleic acid sequences representing the target sequence and gRNA, 19, 20, 21, 22, or more bases can pair complementary to form a complex.
  • the direct repeat sequence has at least 90% sequence identity to any one of SEQ ID Nos. 13-16. In some embodiments, the direct repeat sequence has one or more base substitutions, deletions or additions (for example, 1, 2, 3 , 4, 5, 6, 7, 8, 9 or 10 base substitutions, deletions or additions).
  • the direct repeat sequence is as shown in any one of SEQ ID No. 13-16.
  • gRNA Guide RNA
  • the present invention provides a gRNA, which includes a first segment and a second segment; the first segment is also called “skeleton region”, “protein binding segment”, “protein binding sequence”, or “Direct Repeat (Direct Repeat) sequence”; sequence's boot sequence”.
  • the first section of the gRNA can interact with the Cas protein of the present invention, so that the Cas protein and the gRNA form a complex.
  • the first segment is a direct repeat sequence as described above.
  • the nucleic acid-targeting sequence or nucleic acid-targeting segment of the present invention comprises a nucleotide sequence that is complementary to a sequence in the target nucleic acid.
  • the nucleic acid-targeting targeting sequence or nucleic acid-targeting segment of the present invention interacts with the target nucleic acid in a sequence-specific manner through hybridization (ie, base pairing). Accordingly, the nucleic acid-targeting sequence or nucleic acid-targeting segment can be altered, or modified, to hybridize to any desired sequence within the target nucleic acid.
  • the nucleic acid is selected from DNA or RNA.
  • the percent complementarity between the nucleic acid-targeting sequence or nucleic acid-targeting segment and the target nucleic acid target sequence can be at least 60% (e.g., at least 65%, at least 70%, at least 75%, at least 80%) , at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100%).
  • the "framework region”, "protein binding segment”, “protein binding sequence”, or “direct repeat sequence” of gRNA of the present invention can interact with CRISPR protein (or, Cas protein).
  • the gRNA of the present invention guides its interacting Cas protein to the specific nucleotide sequence in the target nucleic acid through the action of the targeting sequence of the nucleic acid.
  • the guide RNA comprises a first segment and a second segment from 5' to 3' direction.
  • the second segment can also be understood as a guide sequence that hybridizes with the target sequence.
  • the gRNA of the present invention can form a complex with the Cas protein.
  • PAM protospacer adjacent motif
  • PAM protospacer adjacent motif
  • PAM protospacer adjacent motif
  • the present invention also provides a carrier, which comprises the above-mentioned Cas protein, isolated nucleic acid molecule or polynucleotide; preferably, it also includes a regulatory element operably linked thereto.
  • the regulatory element is selected from one or more of the following group: enhancer, transposon, promoter, terminator, leader sequence, polyadenylation sequence, marker gene.
  • the vectors include cloning vectors, expression vectors, shuttle vectors, and integration vectors.
  • the vectors included in the system are viral vectors (such as retroviral vectors, lentiviral vectors, adenoviral vectors, adeno-associated vectors, and herpes simplex vectors), and can also be plasmids, viruses, cosmids, Phage and other types, which are well known to those skilled in the art.
  • viral vectors such as retroviral vectors, lentiviral vectors, adenoviral vectors, adeno-associated vectors, and herpes simplex vectors
  • viruses can also be plasmids, viruses, cosmids, Phage and other types, which are well known to those skilled in the art.
  • the present invention provides an engineered non-naturally occurring vector system, or a CRISPR-Cas system, which includes a Cas protein or a nucleic acid sequence encoding the Cas protein and nucleic acid encoding one or more guide RNAs.
  • the nucleic acid sequence encoding the Cas protein and the nucleic acid encoding one or more guide RNAs are artificially synthesized.
  • nucleic acid sequence encoding the Cas protein and the nucleic acid encoding one or more guide RNAs do not co-occur naturally.
  • the one or more guide RNAs target one or more target sequences in the cell.
  • the one or more target sequences hybridize to the genomic locus of the DNA molecule encoding one or more gene products, and guide the Cas protein to the genomic locus of the DNA molecule of the one or more gene products, the Cas protein
  • the target sequence is modified, edited or cleaved upon reaching the position of the target sequence whereby expression of the one or more gene products is altered or modified.
  • the cells of the present invention include one or more of animals, plants or microorganisms.
  • the Cas protein is codon-optimized for expression in cells.
  • the Cas protein cleaves one or both strands at the position of the target sequence.
  • the Cas protein cuts the complementary strand and/or the non-complementary strand of the target nucleic acid under the guidance of gRNA.
  • the Cas protein simultaneously cuts the complementary strand and the non-complementary strand of the target nucleic acid.
  • the gRNA guides the Cas protein to recognize and bind to the complementary strand
  • the non-complementary strand is a nucleic acid strand paired with the complementary strand.
  • the PAM sequence is located on a non-complementary strand that contains a PAM complementary sequence that pairs with the aforementioned PAM sequence.
  • the cleavage site of Cas-sf0005 to the complementary strand of the target sequence is between the 21nt and 22nt of the 5' end of the PAM complementary sequence, and the cleavage site of Cas-sf0005 to the non-complementary strand of the target sequence is at the PAM sequence
  • the gRNA guides the Cas-sf0005 protein to recognize and bind to the above-mentioned complementary strand, and the above-mentioned non-complementary strand is a DNA strand paired with the complementary strand.
  • the cleavage site of Cas-sf9417 to the complementary strand of the target sequence is between the 21nt and 22nt of the 5' end of the PAM complementary sequence, and the cleavage site of Cas-sf9417 to the non-complementary strand of the target sequence is in the PAM sequence
  • the gRNA guides the Cas-sf9417 protein to recognize and bind to the above-mentioned complementary strand, and the above-mentioned non-complementary strand is a DNA strand paired with the complementary strand.
  • the cleavage site of Cas-sf8553 to the complementary strand of the target sequence is between the 21nt and 23nt of the 5' end of the PAM complementary sequence, and the cleavage site of Cas-sf8553 to the non-complementary strand of the target sequence is in the PAM sequence
  • the gRNA guides the Cas-sf8553 protein to recognize and bind to the above-mentioned complementary strand, and the above-mentioned non-complementary strand is a DNA strand paired with the complementary strand.
  • the present invention also provides an engineered non-naturally occurring vector system which may comprise one or more vectors including:
  • components (a) and (b) are located on the same or different carriers of the system.
  • the first and second regulatory elements include promoters (e.g., constitutive or inducible promoters), enhancers (e.g., 35S promoter or 35S enhanced promoter), internal ribosome entry sites (IRES), and other Expression control elements (eg, transcription termination signals, such as polyadenylation signals and polyU sequences).
  • promoters e.g., constitutive or inducible promoters
  • enhancers e.g., 35S promoter or 35S enhanced promoter
  • IVS internal ribosome entry sites
  • Expression control elements eg, transcription termination signals, such as polyadenylation signals and polyU sequences.
  • the vectors in the system are viral vectors (such as retroviral vectors, lentiviral vectors, adenoviral vectors, adeno-associated vectors, and herpes simplex vectors), and can also be plasmids, viruses, cosmids, phage etc., which are well known to those skilled in the art.
  • viral vectors such as retroviral vectors, lentiviral vectors, adenoviral vectors, adeno-associated vectors, and herpes simplex vectors
  • the systems provided herein are in delivery systems.
  • the delivery system is nanoparticles, liposomes, exosomes, microvesicles and gene guns.
  • the target sequence is a DNA or RNA sequence from a prokaryotic or eukaryotic cell. In one embodiment, the target sequence is a non-naturally occurring DNA or RNA sequence.
  • the target sequence is present intracellularly. In one embodiment, the target sequence is present in the nucleus or in the cytoplasm (eg, an organelle). In one embodiment, the cells are eukaryotic cells. In other embodiments, the cells are prokaryotic cells.
  • the Cas protein is linked with one or more NLS sequences.
  • the fusion protein comprises one or more NLS sequences.
  • the NLS sequence is linked to the N- or C-terminus of the protein.
  • the NLS sequence is fused to the N- or C-terminus of the protein.
  • the present invention relates to an engineered CRISPR system comprising the above-mentioned Cas protein and one or more guide RNAs, wherein the guide RNAs include direct repeat sequences and spacers capable of hybridizing with target nucleic acids sequence, the Cas protein can bind the guide RNA and target a target nucleic acid sequence complementary to the spacer sequence.
  • the Cas enzyme is Cas-sf0005
  • the target nucleic acid is DNA (preferably, double-stranded DNA)
  • the target nucleic acid is located at the 3' end of the protospacer adjacent motif (PAM)
  • PAM protospacer adjacent motif
  • the present invention provides a compound or composition comprising:
  • protein component it is selected from: above-mentioned Cas protein, derivatization protein or fusion protein, and any combination thereof;
  • nucleic acid component comprising (a) a guide sequence capable of hybridizing to a target sequence; and (b) a direct repeat sequence capable of combining with the Cas protein of the present invention.
  • the protein component and the nucleic acid component associate with each other to form a complex.
  • the nucleic acid component is a guide RNA in a CRISPR-Cas system.
  • the complex or composition is non-naturally occurring or modified. In one embodiment, at least one component of the complex or composition is non-naturally occurring or modified. In one embodiment, the first component is non-naturally occurring or modified; and/or, the second component is non-naturally occurring or modified.
  • the present invention also provides an activated CRISPR complex, the activated CRISPR complex comprising: (1) protein component, which is selected from: Cas protein, derivatized protein or fusion protein of the present invention, and any combination thereof; (2) gRNA, which comprises (a) a guide sequence capable of hybridizing to a target sequence; and (b) a direct repeat sequence capable of combining with the Cas protein of the present invention; and (3) combined on the gRNA the target sequence.
  • the binding is through the binding of the nucleic acid-targeting targeting sequence on the gRNA to the target nucleic acid.
  • activated CRISPR complex refers to the complex after binding or modification of Cas protein, gRNA and target nucleic acid in the CRISPR system.
  • the Cas protein and gRNA of the present invention can form a binary complex, which is activated when combined with a nucleic acid substrate to form an activated CRISPR complex.
  • the nucleic acid substrate and the spacer sequence in the gRNA (or referred to as , complementary to the guide sequence that hybridizes to the target nucleic acid).
  • the spacer sequence of the gRNA exactly matches the target substrate. In other embodiments, the spacer sequence of the gRNA matches a portion (contiguous or non-contiguous) of the target substrate.
  • the activated CRISPR complex may exhibit side branch nuclease cleavage activity, and the side branch nuclease cleavage activity refers to the non-specific cleavage activity or random cleavage activity for single-stranded nucleic acid exhibited by the activated CRISPR complex.
  • Cutting activity also referred to as trans cutting activity in the art.
  • the Cas protein, gRNA, fusion protein, nucleic acid molecule, carrier, system, complex and composition of the present invention can be delivered by any method known in the art. Such methods include, but are not limited to, electroporation, lipofection, nucleofection, microinjection, sonoporation, biolistic, calcium phosphate-mediated transfection, cationic transfection, lipofection, dendritic Transfection, heat shock transfection, nucleofection, magnetofection, lipofection, punch transfection, optical transfection, reagent-enhanced nucleic acid uptake, and via liposomes, immunoliposomes, virosomes, artificial viruses body delivery.
  • the present invention provides a delivery composition, which comprises a delivery carrier, and one or any several selected from the following: Cas protein, fusion protein, nucleic acid molecule, carrier, system, Compounds and compositions.
  • the delivery vehicle is a particle.
  • the delivery vehicle is selected from lipid particles, sugar particles, metal particles, protein particles, liposomes, exosomes, microvesicles, gene guns, or viral vectors (e.g., replication-deficient retroviruses) , lentivirus, adenovirus, or adeno-associated virus).
  • viral vectors e.g., replication-deficient retroviruses
  • lentivirus e.g., lentivirus, adenovirus, or adeno-associated virus
  • the present invention also relates to an in vitro, ex vivo or in vivo cell or cell line or their progeny, said cell or cell line or their progeny comprising: Cas protein, fusion protein, nucleic acid according to the present invention Molecules, protein-nucleic acid complexes, activated CRISPR complexes, vectors, delivery compositions of the invention.
  • the cells are prokaryotic cells.
  • the cells are eukaryotic cells. In certain embodiments, the cells are mammalian cells. In certain embodiments, the cells are human cells. In certain embodiments, the cell is a non-human mammalian cell, such as a non-human primate, bovine, ovine, porcine, canine, monkey, rabbit, rodent (eg, rat or mouse) cell. In certain embodiments, the cell is a non-mammalian eukaryotic cell, such as a cell of a poultry bird (eg, chicken), fish or crustacean (eg, clam, shrimp).
  • a poultry bird eg, chicken
  • fish or crustacean eg, clam, shrimp
  • the cell is a plant cell, such as a cell possessed by a monocotyledonous or dicotyledonous plant or a cell possessed by a cultivated plant or food crop such as cassava, corn, sorghum, soybean, wheat, oat or rice, such as Algae, trees or produce plants, fruits or vegetables (eg, trees such as citrus trees, nut trees; nightshade, cotton, tobacco, tomato, grape, coffee, cocoa, etc.).
  • a plant cell such as a cell possessed by a monocotyledonous or dicotyledonous plant or a cell possessed by a cultivated plant or food crop such as cassava, corn, sorghum, soybean, wheat, oat or rice, such as Algae, trees or produce plants, fruits or vegetables (eg, trees such as citrus trees, nut trees; nightshade, cotton, tobacco, tomato, grape, coffee, cocoa, etc.).
  • the cells are stem cells or stem cell lines.
  • a host cell of the invention contains a gene or genome modification that is not present in its wild type.
  • the Cas protein, nucleic acid, the above-mentioned composition, the above-mentioned CIRSPR/Cas system, the above-mentioned carrier system, the above-mentioned delivery composition or the above-mentioned activated CRISPR complex or the above-mentioned host cell of the present invention can be used for any or any of the following purposes: targeting and/or edit target nucleic acid; cut double-stranded DNA, single-stranded DNA or single-stranded RNA; non-specifically cut and/or degrade side branch nucleic acid; non-specifically cut single-stranded nucleic acid; nucleic acid detection; detect nucleic acid in target sample; specifically Edit double-stranded nucleic acid; base edit double-stranded nucleic acid; base edit single-stranded nucleic acid. In other embodiments, it can also be used to prepare reagents or kits for any or any of the above purposes.
  • the present invention also provides the application of the above-mentioned Cas protein, nucleic acid, the above-mentioned composition, the above-mentioned CIRSPR/Cas system, the above-mentioned carrier system, the above-mentioned delivery composition or the above-mentioned activated CRISPR complex in gene editing, gene targeting or gene cutting; Or, the use in the preparation of reagents or kits for gene editing, gene targeting or gene cutting.
  • the gene editing, gene targeting or gene cleavage is intracellular and/or extracellular gene editing, gene targeting or gene cleavage.
  • the present invention also provides a method for editing a target nucleic acid, targeting a target nucleic acid or cutting a target nucleic acid, the method comprising combining the target nucleic acid with the above-mentioned Cas protein, nucleic acid, the above-mentioned composition, the above-mentioned CIRSPR/Cas system, the above-mentioned carrier system, The aforementioned delivery composition or the aforementioned activated CRISPR complex is contacted.
  • the method is editing, targeting, or cleaving a target nucleic acid intracellularly or extracellularly.
  • the gene editing or editing target nucleic acid includes modifying genes, knocking out genes, changing the expression of gene products, repairing mutations, and/or inserting polynucleotides, gene mutations.
  • the editing can be performed in prokaryotic cells and/or eukaryotic cells.
  • the present invention also provides the application of the above-mentioned Cas protein, nucleic acid, the above-mentioned composition, the above-mentioned CIRSPR/Cas system, the above-mentioned carrier system, the above-mentioned delivery composition or the above-mentioned activated CRISPR complex in nucleic acid detection, or in the preparation of Use in reagents or kits for nucleic acid detection.
  • the present invention also provides a method for cutting single-stranded nucleic acid, the method comprising, contacting the nucleic acid population with the above-mentioned Cas protein and gRNA, wherein the nucleic acid population comprises a target nucleic acid and a plurality of non-target single-stranded nucleic acids , the Cas protein cleaves the plurality of non-target single-stranded nucleic acids.
  • the gRNA is capable of binding the Cas protein.
  • the gRNA is capable of targeting the target nucleic acid.
  • the contacting can be inside the cell in vitro, ex vivo or in vivo.
  • the cleavage of single-stranded nucleic acid is non-specific cleavage.
  • the present invention also provides the above-mentioned Cas protein, nucleic acid, the above-mentioned composition, the above-mentioned CIRSPR/Cas system, the above-mentioned carrier system, the above-mentioned delivery composition or the above-mentioned activated CRISPR complex in non-specific cutting single-stranded nucleic acid Application, or use in the preparation of reagents or kits for non-specific cutting of single-stranded nucleic acids.
  • the present invention also provides a kit for gene editing, gene targeting or gene cleavage, the kit comprising the above-mentioned Cas protein, gRNA, nucleic acid, the above-mentioned composition, the above-mentioned CIRSPR/Cas system, the above-mentioned The vector system, the delivery composition described above, the activated CRISPR complex described above, or the host cell described above.
  • the present invention also provides a test kit for detecting target nucleic acid in a sample, the test kit comprising: (a) Cas protein, or nucleic acid encoding the Cas protein; (b) guide RNA, or a nucleic acid encoding the guide RNA, or a precursor RNA comprising the guide RNA, or a nucleic acid encoding the precursor RNA; and (c) a single-stranded nucleic acid that is single-stranded and does not hybridize to the guide RNA Detector.
  • precursor RNAs can be cleaved or processed into mature guide RNAs as described above.
  • the invention provides the above-mentioned Cas protein, nucleic acid, the above-mentioned composition, the above-mentioned CIRSPR/Cas system, the above-mentioned carrier system, the above-mentioned delivery composition, the above-mentioned activated CRISPR complex or the above-mentioned host cell in the preparation or kit Purpose, described preparation or test kit are used for:
  • the above-mentioned gene or genome editing is gene or genome editing performed inside or outside the cell.
  • the target nucleic acid detection and/or diagnosis is in vitro target nucleic acid detection and/or diagnosis.
  • the treatment of said disease is the treatment of a condition caused by a defect in a target sequence in a target locus.
  • the present invention provides a method for detecting a target nucleic acid in a sample, the method comprising contacting the sample with the Cas protein, gRNA (guide RNA) and a single-stranded nucleic acid detector, the gRNA including the Cas protein-binding region and guide sequence hybridized with target nucleic acid; detect the detectable signal produced by the Cas protein cleavage single-stranded nucleic acid detector, thereby detecting target nucleic acid; the single-stranded nucleic acid detector does not hybridize with the gRNA .
  • the present invention also provides a method for specifically modifying a target nucleic acid, the method comprising: combining the target nucleic acid with the above-mentioned Cas protein, nucleic acid, the above-mentioned composition, the above-mentioned CIRSPR/Cas system, the above-mentioned carrier system, and the above-mentioned delivery composition or the above-mentioned activated CRISPR complex contacts.
  • This specific modification can occur in vivo or in vitro.
  • This specific modification can occur intracellularly or extracellularly.
  • the cells are selected from prokaryotic or eukaryotic cells, eg, animal cells, plant cells, or microbial cells.
  • the modification refers to a break in the target sequence, such as a single-strand/double-strand break in DNA, or a single-strand break in RNA.
  • the method further comprises contacting the target nucleic acid with a donor polynucleotide, wherein the donor polynucleotide, a portion of the donor polynucleotide, a copy of the donor polynucleotide, or the donor polynucleotide Part of the copy of the target nucleic acid is integrated.
  • said modifying further comprises inserting an editing template (eg, exogenous nucleic acid) into said break.
  • an editing template eg, exogenous nucleic acid
  • the method further comprises: contacting the editing template with the target nucleic acid, or delivering it to a cell comprising the target nucleic acid.
  • the method repairs the disrupted target gene by homologous recombination with an exogenous template polynucleotide; in some embodiments, the repair results in a mutation comprising one or Insertion, deletion, or substitution of multiple nucleotides, in other embodiments, the mutation results in one or more amino acid changes in a protein expressed from a gene comprising the target sequence.
  • the present invention provides a method for detecting target nucleic acid in a sample, the method comprising combining the sample with the above-mentioned Cas protein, nucleic acid, the above-mentioned composition, the above-mentioned CIRSPR/Cas system, the above-mentioned carrier system, the above-mentioned delivery composition or
  • the above-mentioned activated CRISPR complex is in contact with the single-stranded nucleic acid detector; the detectable signal generated by the Cas protein cutting the single-stranded nucleic acid detector is detected, thereby detecting the target nucleic acid.
  • the target nucleic acid includes ribonucleotide or deoxyribonucleotide; includes single-stranded nucleic acid, double-stranded nucleic acid, such as single-stranded DNA, double-stranded DNA, single-stranded RNA, double-stranded RNA.
  • the target nucleic acid is derived from samples such as viruses, bacteria, microorganisms, soil, water sources, human bodies, animals, and plants.
  • the target nucleic acid is a product enriched or amplified by PCR, NASBA, RPA, SDA, LAMP, HAD, NEAR, MDA, RCA, LCR, RAM and other methods.
  • the target nucleic acid is a viral nucleic acid, a bacterial nucleic acid, a specific nucleic acid associated with a disease, such as a specific mutation site or a SNP site or a nucleic acid that is different from a control;
  • the virus is a plant Viruses or animal viruses, for example, papillomaviruses, hepadnaviruses, herpesviruses, adenoviruses, poxviruses, parvoviruses, coronaviruses; preferably, the viruses are coronaviruses, preferably SARS, SARS-CoV2 (COVID-19) -19), HCoV-229E, HCoV-OC43, HCoV-NL63, HCoV-HKU1, Mers-Cov.
  • the gRNA has at least 50% matching degree with the target sequence on the target nucleic acid, preferably at least 60%, preferably at least 70%, preferably at least 80%, preferably at least 90%.
  • the characteristic sites completely match the gRNA.
  • the detection method may comprise one or more gRNAs with different guide sequences, which target different target sequences.
  • the single-stranded nucleic acid detectors include but are not limited to single-stranded DNA, single-stranded RNA, DNA-RNA hybrids, nucleic acid analogs, base modifiers, and single-stranded nucleic acid detection containing abasic spacers
  • Nucleic acid analogs include but are not limited to: locked nucleic acid, bridge nucleic acid, morpholine nucleic acid, ethylene glycol nucleic acid, hexitol nucleic acid, threose nucleic acid, arabinose nucleic acid, 2'oxymethyl RNA, 2' Methoxyacetyl RNA, 2' fluoro RNA, 2' amino RNA, 4' thio RNA and combinations thereof, including optional ribonucleotide or deoxyribonucleotide residues.
  • the detectable signal is realized by the following methods: vision-based detection, sensor-based detection, color detection, fluorescence signal-based detection, gold nanoparticle-based detection, fluorescence polarization, colloidal phase transition/dispersion, electrical Chemical detection and semiconductor-based detection.
  • the two ends of the single-stranded nucleic acid detector are respectively provided with a fluorescent group and a quencher group, and when the single-stranded nucleic acid detector is cleaved, it can display a detectable fluorescent signal.
  • the fluorescent group is selected from one or any of FAM, FITC, VIC, JOE, TET, CY3, CY5, ROX, Texas Red or LC RED460; the quenching group is selected from BHQ1, BHQ2, BHQ3 , Dabcy1 or Tamra or any of several.
  • the 5' end and the 3' end of the single-stranded nucleic acid detector are respectively provided with different labeling molecules, and are detected by means of colloidal gold detection before and after the single-stranded nucleic acid detector is cleaved by the Cas protein.
  • Colloidal gold test results after being cleaved by Cas protein; the single-stranded nucleic acid detector will show different color development results on the detection line and quality control line of colloidal gold before being cleaved by Cas protein and after being cleaved by Cas protein.
  • the method of detecting a target nucleic acid can further comprise comparing the level of the detectable signal to a reference signal level, and determining the amount of the target nucleic acid in the sample based on the level of the detectable signal.
  • the method for detecting target nucleic acid can also include using RNA reporter nucleic acid and DNA reporter nucleic acid (for example, fluorescent color) on different channels, and by measuring the signal level of RNA and DNA reporter molecules, and by measuring The amount of target nucleic acid in the RNA and DNA reporter molecules is used to determine the level of detectable signal, and the level of detectable signal is sampled based on the combined (eg, using minimum or product) detectable signal levels.
  • RNA reporter nucleic acid and DNA reporter nucleic acid for example, fluorescent color
  • the target gene is present in the cell.
  • the cells are prokaryotic cells.
  • the cells are eukaryotic cells.
  • the cells are animal cells.
  • the cells are human cells.
  • the cell is a plant cell, for example a cell possessed by a cultivated plant (such as cassava, corn, sorghum, wheat or rice), algae, tree or vegetable.
  • a cultivated plant such as cassava, corn, sorghum, wheat or rice
  • algae such as tree or vegetable.
  • the target gene is present in a nucleic acid molecule (eg, a plasmid) in vitro.
  • a nucleic acid molecule eg, a plasmid
  • the target gene is present on a plasmid.
  • Cas protein, Cas enzyme, and Cas effector protein can be used interchangeably;
  • the inventor has discovered and identified a Cas effector protein for the first time, which has an amino acid sequence selected from the following:
  • (iii) have at least 20%, at least 30%, at least 40%, at least 50%, at least 60% of the sequence shown in SEQ ID No.1 or SEQ ID No.4 or SEQ ID No.7 or SEQ ID No.10 %, at least 70%, at least 80%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, Sequences having at least 98%, or at least 99%, sequence identity.
  • Nucleic acid cutting or cleavage nucleic acid herein include: DNA or RNA breakage (Cis cleavage), DNA or RNA in target nucleic acid produced by Cas enzyme described herein (ie non-specific or non-targeted, Trans cleavage).
  • the cleavage is a double-strand DNA break.
  • the cleavage is a single stranded DNA break or a single stranded RNA break.
  • CRISPR-CRISPR-associated (Cas) system or “CRISPR system” is used interchangeably and has the skill in the art The meaning generally understood by personnel, which generally comprises transcription products or other elements related to the expression of CRISPR-associated (“Cas”) genes, or transcription products or other elements capable of directing the activity of said Cas genes.
  • CRISPR/Cas complex refers to a complex formed by the combination of guide RNA (guide RNA) or mature crRNA and the Cas protein, which comprises a guide sequence that hybridizes to the target sequence and binds to the The direct repeat sequence bound by the Cas protein, the complex can recognize and cut polynucleotides that can hybridize with the guide RNA or mature crRNA.
  • Guide RNA guide RNA, gRNA
  • RNA guide RNA, gRNA
  • matrix crRNA target RNA
  • guide sequence RNA sequence
  • a guide RNA can comprise, or consist essentially of, or consist of, a direct repeat and a guide sequence.
  • a guide sequence is any polynucleotide sequence that is sufficiently complementary to a target sequence to hybridize to and direct specific binding of the CRISPR/Cas complex to the target sequence.
  • the degree of complementarity between the guide sequence and its corresponding target sequence is at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or At least 99%. Determining the optimal alignment is within the ability of one of ordinary skill in the art. For example, there are public and commercially available alignment algorithms and programs such as, but not limited to, Clustal W, Smith-Waterman in matlab, Bowtie, Geneious, Biopython, and SeqMan.
  • Target sequence refers to a polynucleotide targeted by a guide sequence in a gRNA, such as a sequence that is complementary to the guide sequence, wherein hybridization between the target sequence and the guide sequence will facilitate the CRISPR/Cas complex (including Cas protein and gRNA) formation. Perfect complementarity is not required, so long as there is sufficient complementarity to cause hybridization and promote the formation of a CRISPR/Cas complex.
  • a target sequence can comprise any polynucleotide, such as DNA or RNA.
  • the target sequence is intracellular or extracellular.
  • the target sequence is located in the nucleus or cytoplasm of the cell.
  • the target sequence may be located in an organelle of the eukaryotic cell such as the mitochondria or chloroplast.
  • a sequence or template that can be used for recombination into a target locus comprising the target sequence is referred to as an "editing template" or "editing polynucleotide” or “editing sequence”.
  • the editing template is an exogenous nucleic acid.
  • the recombination is homologous recombination.
  • a "target sequence” or “target polynucleotide” or “target nucleic acid” may be any polynucleotide endogenous or exogenous to a cell (eg, a eukaryotic cell).
  • the target polynucleotide can be a polynucleotide present in the nucleus of a eukaryotic cell.
  • the target polynucleotide can be a sequence encoding a gene product (eg, protein) or a non-coding sequence (eg, regulatory polynucleotide or dummy DNA).
  • the target sequence should be related to a protospacer adjacent motif (PAM).
  • PAM protospacer adjacent motif
  • the single-stranded nucleic acid detector of the present invention refers to a sequence containing 2-200 nucleotides, preferably, has 2-150 nucleotides, preferably, 3-100 nucleotides, preferably, 3-30 nucleotides Nucleotides, preferably, 4-20 nucleotides, more preferably, 5-15 nucleotides. It is preferably a single-stranded DNA molecule, a single-stranded RNA molecule or a single-stranded DNA-RNA hybrid.
  • the two ends of the single-stranded nucleic acid detector include different reporter groups or marker molecules. When it is in the initial state (that is, when it is not cut), it does not show a reporter signal. When the single-stranded nucleic acid detector is cut, it shows A detectable signal is shown, that is, a detectable difference is shown after cleavage from before cleavage.
  • the reporter group or marker molecule includes a fluorescent group and a quencher group
  • the fluorescent group is selected from FAM, FITC, VIC, JOE, TET, CY3, CY5, ROX, Texas Red Or one or any several in LC RED460; Described quenching group is selected from one or any several in BHQ1, BHQ2, BHQ3, Dabcy1 or Tamra.
  • the single-stranded nucleic acid detector has a first molecule (such as FAM or FITC) connected to the 5' end and a second molecule (such as biotin) connected to the 3' end.
  • the reaction system containing a single-stranded nucleic acid detector cooperates with a flow strip to detect target nucleic acid (preferably, colloidal gold detection method).
  • the flow strip is designed to have two capture lines, the sample contact end (colloidal gold) is provided with an antibody binding to the first molecule (i.e. the first molecule antibody), and the first line (control line) contains the binding second molecule antibody.
  • An antibody of one molecule of antibody contains a second molecule of antibody (ie, a second molecule of antibody, such as avidin) that binds to the second molecule at the second line (test line).
  • a second molecule of antibody such as avidin
  • the first molecule of antibody binds to the first molecule carrying the cleaved or uncleaved oligonucleotide to the capture line
  • the cleaved reporter will bind the first molecule of antibody at the first capture line antibody
  • the uncleaved reporter will bind a second molecule of antibody at the second capture line.
  • the incorporation of reporter groups at each line will result in a strong readout/signal (eg color).
  • the invention relates to the use of a flow strip as described herein for the detection of nucleic acids.
  • the invention relates to a method for the detection of nucleic acids using a flow strip as defined herein, such as a (lateral) flow test or a (lateral) flow immunochromatographic assay.
  • the molecules in the single-stranded nucleic acid detector can be replaced with each other, or the position of the molecules can be changed, as long as the reporting principle is the same or similar to the present invention, the improved methods are also included in the present invention.
  • the detection method of the present invention can be used for the quantitative detection of the target nucleic acid to be detected.
  • the quantitative detection index can be quantified according to the signal intensity of the reporter group, such as according to the luminescent intensity of the fluorescent group, or according to the width of the color band.
  • wild type has the meaning generally understood by those skilled in the art, which means a typical form of an organism, bacterial strain, gene or a characteristic that is different from a mutant or variant form when it exists in nature, It can be isolated from its source in nature and has not been intentionally modified by man.
  • the term "derivatization” refers to a chemical modification of an amino acid, polypeptide or protein to which one or more substituents have been covalently attached. Substituents may also be referred to as side chains.
  • a derivatized protein is a derivative of the protein, and typically, the derivatization of the protein does not adversely affect the desired activity of the protein (e.g., binding activity to a guide RNA, endonuclease activity, binding to a target sequence under the guidance of a guide RNA).
  • specific site binding and cleavage activity that is to say, the derivative of the protein has the same activity as the protein.
  • protein derivative refers to a modified form of a protein, for example, wherein one or more amino acids of the protein can be deleted, inserted, modified and/or substituted.
  • nucleic acid molecule or polypeptide when used to describe a nucleic acid molecule or polypeptide, mean that the nucleic acid molecule or polypeptide is at least substantially free from at least one other component with which they exist in nature or are associated with them as found in nature.
  • an "ortholog" of a protein as described herein refers to a protein belonging to a different species that performs the same or a similar function as the protein that is its ortholog.
  • identity is used to refer to the match of sequences between two polypeptides or between two nucleic acids.
  • a position in both sequences being compared is occupied by the same base or amino acid monomer subunit (for example, a position in each of the two DNA molecules is occupied by an adenine, or both a position in each of the polypeptides is occupied by lysine)
  • Percent identity between two sequences is a function of the number of matching positions shared by the two sequences divided by the number of positions being compared x 100. For example, two sequences are 60% identical if 6 out of 10 positions match.
  • the DNA sequences CTGACT and CAGGTT share 50% identity (3 out of a total of 6 positions match).
  • comparisons are made when two sequences are aligned for maximum identity.
  • Such alignments can be achieved using, for example, the method of Needleman et al. (1970) J. Mol. Biol. 48:443-453 which can be conveniently performed by computer programs such as the Align program (DNAstar, Inc.).
  • the algorithm of E. Meyers and W. Miller (Comput. Appl Biosci., 4:11-17 (1988)), which has been integrated into the ALIGN program (version 2.0), can also be used, using the PAM120 weight residue table , a gap length penalty of 12, and a gap penalty of 4 to determine the percent identity between two amino acid sequences.
  • the algorithm of Needleman and Wunsch (J MoI Biol. 48:444-453 (1970)) in the GAP program that has been incorporated into the GCG software package can be used, using the Blossum 62 matrix or PAM250 matrix with a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6 to determine percent identity between two amino acid sequences .
  • vector refers to a nucleic acid molecule capable of transporting another nucleic acid molecule to which it has been linked.
  • Vectors include, but are not limited to, nucleic acid molecules that are single-stranded, double-stranded, or partially double-stranded; nucleic acid molecules that include one or more free ends, no free ends (e.g., circular); nucleic acid molecules that include DNA, RNA, or both. nucleic acid molecules; and other miscellaneous polynucleotides known in the art.
  • a vector can be introduced into a host cell by transformation, transduction or transfection, so that the genetic material elements it carries can be expressed in the host cell.
  • a vector can be introduced into a host cell to thereby produce transcripts, proteins, or peptides, including proteins, fusion proteins, isolated nucleic acid molecules, etc., as described herein (e.g., CRISPR transcripts, such as nucleic acid transcripts , protein, or enzyme).
  • a vector can contain a variety of elements that control expression, including but not limited to, promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes.
  • the vector may also contain an origin of replication.
  • Plasmid refers to a circular double-stranded DNA loop into which additional DNA segments can be inserted, eg, by standard molecular cloning techniques.
  • viral vector in which virally derived DNA or RNA sequences are present in a virus used for packaging (e.g., retroviruses, replication defective retroviruses, adenoviruses, replication defective adenoviruses, and adeno-associated virus) vector.
  • virus used for packaging e.g., retroviruses, replication defective retroviruses, adenoviruses, replication defective adenoviruses, and adeno-associated virus
  • Viral vectors also comprise polynucleotides carried by the virus used for transfection into a host cell.
  • Certain vectors eg, bacterial vectors with a bacterial origin of replication and episomal mammalian vectors
  • vectors eg, non-episomal mammalian vectors
  • certain vectors are capable of directing the expression of genes to which they are operably linked. Such vectors are referred to herein as "expression vectors.”
  • the term "host cell” refers to cells that can be used to introduce vectors, including, but not limited to, prokaryotic cells such as Escherichia coli or Bacillus subtilis, such as microbial cells, fungal cells, animal cells, and plants Cells of eukaryotic cells.
  • the design of the expression vector may depend on factors such as the choice of host cell to be transformed, the level of expression desired, and the like.
  • regulatory element is intended to include promoters, enhancers, internal ribosomal entry sites (IRES), and other expression control elements (e.g., transcription termination signals, such as polyadenylation signals and Poly U sequence), its detailed description can refer to Godel (Goeddel), "Gene Expression Technology: Methods of Enzyme” (GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY) 185, Academic Press (Academic Press), San Diego (San Diego) , California (1990).
  • IRES internal ribosomal entry sites
  • regulatory elements include those that direct the constitutive expression of a nucleotide sequence in many types of host cells as well as those that direct the expression of the nucleotide sequence only in certain host cells (e.g., tissue-specific regulatory sequences).
  • tissue-specific regulatory sequences may primarily direct expression in a desired tissue of interest, such as muscle, neurons, bone, skin, blood, a specific organ (e.g., liver, pancreas), or a particular cell type (e.g., lymphocytes).
  • regulatory elements may also direct expression in a timing-dependent manner (eg, in a cell cycle-dependent or developmental stage-dependent manner), which may or may not be tissue or cell type specific.
  • the term "regulatory element” encompasses enhancer elements such as WPRE; CMV enhancer; the R-U5' fragment in the LTR of HTLV-1 ((Mol. Cell. Biol., 8( 1), pp. 466-472, 1988); the SV40 enhancer; and the intron sequence between exons 2 and 3 of rabbit ⁇ -globin (Proc.Natl.Acad.Sci.USA., Vol. 78(3), pp. 1527-31, 1981).
  • enhancer elements such as WPRE; CMV enhancer; the R-U5' fragment in the LTR of HTLV-1 ((Mol. Cell. Biol., 8( 1), pp. 466-472, 1988); the SV40 enhancer; and the intron sequence between exons 2 and 3 of rabbit ⁇ -globin (Proc.Natl.Acad.Sci.USA., Vol. 78(3), pp. 1527-31, 1981).
  • promoter has the meaning known to those skilled in the art, which refers to a non-coding nucleotide sequence located upstream of a gene that can promote the expression of a downstream gene.
  • a constitutive promoter is a nucleotide sequence that, when operably linked to a polynucleotide that encodes or defines a gene product, results in the expression of the gene product in the cell under most or all physiological conditions of the cell. generation.
  • An inducible promoter is a nucleotide sequence which, when operably linked to a polynucleotide encoding or defining a gene product, results in a The gene product is produced intracellularly.
  • a tissue-specific promoter is a nucleotide sequence that, when operably linked to a polynucleotide that encodes or defines a gene product, results in a The gene product is produced in the cell.
  • a “nuclear localization signal” or “nuclear localization sequence” is an amino acid sequence that "tags" a protein for import into the nucleus by nuclear transport, ie, a protein with a NLS is transported to the nucleus.
  • NLSs typically contain positively charged Lys or Arg residues exposed on the protein surface.
  • Exemplary nuclear localization sequences include, but are not limited to, NLS from: SV40 large T antigen, EGL-13, c-Myc, and TUS proteins.
  • the NLS comprises a PKKKRKV sequence.
  • the NLS comprises the sequence AVKRPAATKKAGQAKKKKLD.
  • the NLS comprises the sequence PAAKRVKLD.
  • the NLS comprises the sequence MSRRRKANPTKLSENAKKLAKEVEN. In some embodiments, the NLS comprises the KLKIKRPVK sequence.
  • Other nuclear localization sequences include, but are not limited to, the acidic M9 domain of hnRNP A1, the sequences KIPIK and PY-NLS in the yeast transcriptional repressor Mat ⁇ 2.
  • operably linked is intended to mean that the nucleotide sequence of interest is linked to the one or more regulatory elements in a manner that allows expression of the nucleotide sequence (e.g. , in an in vitro transcription/translation system or when the vector is introduced into the host cell, in the host cell).
  • complementarity refers to the ability of a nucleic acid to form one or more hydrogen bonds with another nucleic acid sequence by means of traditional Watson-Crick or other non-traditional types. Percent complementarity represents the percentage of residues in a nucleic acid molecule that can form hydrogen bonds (e.g., Watson-Crick base pairing) with a second nucleic acid sequence (e.g., 5, 6, 7, 8 out of 10 , 9, 10 are 50%, 60%, 70%, 80%, 90%, and 100% complementary). "Perfectly complementary” means that all contiguous residues of one nucleic acid sequence form hydrogen bonds with the same number of contiguous residues in a second nucleic acid sequence.
  • Substantially complementary refers to a group having At least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98 over a region of 30, 35, 40, 45, 50 or more nucleotides %, 99%, or 100% degree of complementarity, or refers to two nucleic acids that hybridize under stringent conditions.
  • stringent conditions for hybridization refer to conditions under which a nucleic acid having complementarity to a target sequence hybridizes primarily to the target sequence and does not substantially hybridize to non-target sequences. Stringent conditions are generally sequence-dependent and vary depending on many factors. In general, the longer the sequence, the higher the temperature at which the sequence will specifically hybridize to its target sequence.
  • hybridizes or “complementary” or “substantially complementary” means that a nucleic acid (e.g. RNA, DNA) comprises a nucleotide sequence that enables it to bind non-covalently, i.e. in a sequence-specific, antiparallel fashion ( That is, a nucleic acid that specifically binds to a complementary nucleic acid) forms base pairs and/or G/U base pairs with another nucleic acid, "anneals" or “hybridizes”.
  • a nucleic acid e.g. RNA, DNA
  • anneals or “hybridizes”.
  • Hybridization requires that the two nucleic acids contain complementary sequences, although there may be mismatches between the bases. Suitable conditions for hybridization between two nucleic acids depend on the length of the nucleic acids and the degree of complementarity, variables well known in the art. Typically, hybridizable nucleic acids are 8 nucleotides or more in length (e.g., 10 nucleotides or more, 12 nucleotides or more, 15 nucleotides or more, 20 nuclei nucleotides or more, 22 nucleotides or more, 25 nucleotides or more, or 30 nucleotides or more).
  • sequence of a polynucleotide need not be 100% complementary to the sequence of its target nucleic acid to specifically hybridize.
  • the polynucleotide may comprise 60% or greater, 65% or greater, 70% or greater, 75% or greater, 80% or greater, 85% or greater, 90% or greater, 95% or Higher, 98% or higher, 99% or higher, 99.5% or higher, or 100% sequence complementarity to a target region in a target nucleic acid sequence to which it hybridizes.
  • Hybridization of the target sequence to the gRNA represents at least 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96% of the nucleic acid sequence of the target sequence and the gRNA , 97%, 98%, 99% or 100% can hybridize to form a complex; or there are at least 12, 15, 16, 17, 18, 19, 20 nucleic acid sequences representing the target sequence and gRNA 1, 21, 22 or more bases can be complementary paired and hybridized to form a complex.
  • the term "expression” refers to the process whereby a polynucleotide is transcribed from a DNA template (such as into mRNA or other RNA transcript) and/or the process whereby the transcribed mRNA is subsequently translated into a peptide, Polypeptide or protein process. Transcripts and encoded polypeptides may be collectively referred to as "gene products.” If the polynucleotide is derived from genomic DNA, expression can include splicing of the mRNA in eukaryotic cells.
  • linker refers to a linear polypeptide formed by linking multiple amino acid residues through peptide bonds.
  • the linker of the present invention may be an artificially synthesized amino acid sequence, or a naturally occurring polypeptide sequence, such as a polypeptide having a hinge region function.
  • Such linker polypeptides are well known in the art (see, e.g., Holliger, P. et al. (1993) Proc. Natl. Acad. Sci. USA 90:6444-6448; Poljak, R.J. et al. (1994) Structure 2:1121- 1123).
  • treating refers to treating or curing a disorder, delaying the onset of symptoms of a disorder, and/or delaying the progression of a disorder.
  • the term "subject” includes, but is not limited to, various animals, plants and microorganisms.
  • mammals such as bovines, equines, ovines, porcines, canines, felines, lagomorphs, rodents (e.g., mice or rats), non-human primates animals (for example, macaques or cynomolgus monkeys) or humans.
  • the subject eg, a human
  • has a disorder eg, a disorder caused by a defect in a disease-associated gene.
  • plant is to be understood as any differentiated multicellular organism capable of photosynthesis, including crop plants at any stage of maturity or development, in particular monocotyledonous or dicotyledonous plants, vegetable crops, including artichokes, kohlrabi, Arugula, leeks, asparagus, lettuce (e.g., head lettuce, leaf lettuce, romaine), bok choy, yellow taro, melons (e.g., melon, watermelon, crenshaw ), cantaloupe, cantaloupe), canola crops (e.g., Brussels sprouts, cabbage, cauliflower, broccoli, kale, kale, Chinese cabbage, bok choy), cardoons, carrots, napa, Okra, onions, celery, parsley, chickpeas, parsnips, endive, peppers, potatoes, gourds (e.g., zucchini, cucumbers, zucchini, squash, squash), radishes, dried bulbs, rutabaga, Purple eggplant (also known as eggplant), sals
  • the present invention has discovered a novel Cas enzyme, and the results of Blast show that the Cas enzyme of the present application is less consistent with the reported Cas enzyme, and it can exhibit nuclease activity in vivo and in vitro, and has broad application prospects .
  • Figure 8 Sequencing results of target genes edited by Cas-sf9417 in eukaryotic cells.
  • Fig. 15 Fluorescence result graph of Cas-sf1846 for nucleic acid detection.
  • the inventors analyzed the metagenomics of uncultured cultures, and identified 4 new Cas enzymes through de-redundancy and protein clustering analysis. Blast results showed that the sequence identity of the Cas protein with the reported Cas protein was low, and the phylogenetic tree analysis showed that it belonged to Similar proteins are named Cas-sf0005, Cas-sf9417, Cas-sf8553 and Cas-sf1846 in the present invention.
  • amino acid sequence, coding nucleic acid sequence and human codon-optimized nucleic acid sequence of the above protein are as follows:
  • the direct repeat sequence of the gRNA corresponding to the Cas-sf0005 protein is CCGUCAACGUUCAACGCUUGCUCGGUUCGCCGAGAC (SEQ ID No.13) or CCGUCAACGUUCAACGCUUGCUCGGUACGCCGAGAC;
  • the direct repeat sequence of the gRNA corresponding to the Cas-sf9417 protein is GUGGGAACCCUUCCUGAUGGCUCGAUCCGUCGAGAC (SEQ ID No.14);
  • the direct repeat sequence of the gRNA corresponding to the Cas-sf8553 protein is GUGAGGACGACAACCGAAGGCUCGGUGCGCCGAGAC (SEQ ID No.15);
  • the direct repeat sequence of the gRNA corresponding to the Cas-sf1846 protein is GUUGCAAGCCUUACUGAUUGCUCCUCACGAGGAGAC (SEQ ID No.16).
  • the PAM library was constructed, and the synthetic sequence was: CGTGTTTCGTAAAGTCTGGAAACGCGGAAGCCCCCAGCGCTTCAGCGTTCNNNNNN TCCCCTACGTGCTGCTGAAGTTGC CCGCAA, N is a random deoxynucleotide, and the underline is the target sequence. After filling in with Klenow enzyme, it was connected into the pacyc184 vector. After transforming Escherichia coli, the plasmid was extracted to form a PAM library.
  • the gRNA Cas-sf0005-5'spacer1 20bp sequence is: CCGUCAACGUUCAACGCUUGCUCGGUUCGCCGAGAC UCCCCUACGUGCUGCUGAAG (the underlined region is the targeting region);
  • TK-117 CGGCATTCCTGCTGAACCGCTCTTCCGATCT;
  • TK-111 GATCGGAAGAGCGGTTCAGCAGGAATGCCG;
  • PAM-after-F ACTCAGGGGTCTTCGGTTTCCGTGTT;
  • Cas-sf0005 protein preference PAM library 50nM Cas-sf0005 protein, 50nM gRNA in buffer Tris-HAC 40mM, Mg(AC)2 30mM, BSA 120 ⁇ g/ml, DTT 12mM, pH7.0, incubate at 25°C for 10min. Add PAM library plasmid (10ng/ ⁇ L), incubate at 37°C for 1h; incubate at 85°C for 20min.
  • the PCR product was subjected to next-generation sequencing, and the PAM sequence was obtained after analysis.
  • the cleaving activity of Cas-sf0005 protein on double-stranded DNA was tested by in vitro detection.
  • gRNA that can be paired with the target nucleic acid is used to guide the Cas-protein to recognize and bind to the double-stranded target nucleic acid; then, the Cas protein stimulates the cleavage activity on the double-stranded target nucleic acid, thereby cutting the double-stranded target nucleic acid in the system.
  • the cleaved double-stranded target nucleic acid is detected by agarose gel electrophoresis.
  • the target nucleic acid is selected as double-stranded DNA, and its sequence is: GAACGCTGAAGCGCTGGGGGCATTA TCCCCTACGTGCTGCTGAAG TTGC into the vector T-Vector-pEASY-Blunt Simple Cloning Vector; TTA in italics is the PAM sequence, and the underlined area is the targeting region.
  • gRNA-Cas-sf0005-5'spacer1-20bp CCGUCAACGUUCAACGCUUGCUCGGUUCGCCGAGAC UCCCCUACGUGCUGCU GAAG (the underlined region is the target region); at the same time, the above fragment was amplified by PCR and used as the target nucleic acid detection; the following reaction system was used: 20 ⁇ L system, Tris-HAC 40mM, Mg(AC)2 30mM, BSA 120 ⁇ g/ml, DTT 12mM, pH7.0, Cas-sf0005 50nM, gRNA 500nM, the final concentration of double-stranded target nucleic acid was 5ng/ ⁇ L.
  • lane 1 is the experimental group
  • lane 2 is the control group
  • lane 3 is the Trans2K plus DNA marker.
  • the Cas-sf0005 in the experimental group can effectively cut the double-stranded nucleic acid in the system when it contains the target nucleic acid (plasmid) and gRNA.
  • lane 1 is the Trans2K plus DNA marker
  • lanes 2-5 are the experimental groups with different PAMs
  • lane 3 is the control group.
  • the Cas-sf0005 in the experimental group can effectively cut the double-stranded nucleic acid in the system when it contains the target nucleic acid (PCR product) of TTN PAM and gRNA.
  • Experimental results show that Cas-sf0005 can be used for cleavage and editing of double-stranded target nucleic acid.
  • the Cas-sf0005 protein was used to verify its gene editing activity in animal cells, and the target was designed for the FUT8 gene of Chinese hamster ovary cells (CHO).
  • the vector pcDNA3.3 was transformed with EGFP fluorescent protein and PuroR resistance gene. Insert the SV40NLS-Cas-sf0005 fusion protein through the restriction site BsmB1; insert the U6 promoter and gRNA sequence through the restriction site Mfe1.
  • the CMV promoter promotes the expression of the fusion protein SV40NLS-Cas-sf0005-NLS-GFP.
  • the protein Cas-sf0005-NLS is connected to the protein GFP with the connecting peptide T2A.
  • the promoter EF-1 ⁇ drives the expression of the puromycin resistance gene.
  • Plating CHO cells are plated to a confluence of 70-80%, and the number of cells seeded in a 12-well plate is 8*10 ⁇ 4 cells/well.
  • Transfection Plate for 6-8 hours for transfection, add 3.25 ⁇ l Lipo3000 to 125 ⁇ l opti-MEM and mix well; add 3 ug plasmid and 10 ⁇ l P3000 to 125 ⁇ l opti-MEM, mix well. The diluted Lipo3000 was mixed evenly with the diluted plasmid, and incubated at room temperature for 5 minutes. The incubated mixture was added to the culture medium on which the cells were layered for transfection.
  • puromycin for selection add puromycin 24 hours after transfection, the final concentration is 10ng/ml. Puromycin was treated for 24 hours and replaced with normal medium to continue culturing for 24 hours.
  • Genomic DNA extraction, PCR amplification near the edited region, sending to hiTOM for sequencing the cells were collected after trypsinization, and the genomic DNA was extracted with the Cell/Tissue Genomic DNA Extraction Kit (Bitec). Genomic DNA was amplified by primers NAFUT8-JC-HITOM-1F1: GGAGTGAGTACGGTGTGCTCCTCCTTACTTACCCTTGG; NAFUT8-JC-HITOM-1R1: GAGTTGGATGCTGGATGGTTGTTCTTTGGTGGGACTATG to amplify the region near the target. PCR products were subjected to hiTOM sequencing (http://121.40.237.174/HiTOM/Sample_acceptance_sanyang.php).
  • the sequencing data was analyzed, and the types and ratios of sequences within the range of 15 nt upstream and 10 nt downstream of the target position were counted, and the editing efficiency of the target position by the Cas-sf0005 protein was obtained.
  • CHO cell FUT8 gene target sequence gR1-FUT8: TTT GACAAACTGGGATACCCACCAC A , the italic part is the PAM sequence, and the underlined area is the targeting region.
  • the gRNA sequence is GCCGUCAACGUUCAACGCUUGCUCGGUUCGCCGAGACGACAAACUGGGAUACCCACCACA, and the underlined region is the targeting region.
  • the cleavage efficiency of Cas-sf0005 on the target nucleotide (double-stranded DNA) complementary strand (TS) and non-complementary strand (NTS) is detected in vitro.
  • 5'6-FAM marks the non-complementary strand (NTS)
  • 5'ROX marks the complementary strand (TS)
  • gRNA guides the Cas-sf0005 protein to recognize and bind to the target nucleic acid complementary strand (TS), thereby cutting the target nucleic acid in the system Complementary strand (TS) and non-complementary strand (NTS)
  • the cleaved target nucleic acid is detected by capillary electrophoresis (ABI 3730xl genetic analyzer).
  • the DNA fragments migrate from the cathode to the anode in the gel, and are arranged according to the length of the fragments.
  • the fluorescent dye When they migrate to the scanning window of the laser scanner at the anode, the fluorescent dye is excited and emits light of a certain wavelength, which is recorded according to the fluorescence intensity.
  • the electrophoretic traces of DNA fragments with fluorescent dyes are recorded according to the actual time when they pass through the laser scanning window, and each fragment is represented by a fluorescence absorption peak. The higher the peak value, the more fragments there are; the peak appearance time is directly related to the fragment size, the smaller the fragment, the earlier the peak appears.
  • the Cas-sf0005 uncut NTS fragment in the FAM fluorescence channel is 380nt, and the Cas-sf0005 cut NTS fragment is about 126nt.
  • the Cas-sf0005 uncut TS fragment size in the ROX fluorescence channel is 380nt, and the Cas-sf0005 cut TS fragment is about 254nt.
  • Fragment cutting efficiency calculation formula: cutting efficiency cut peak area/(cut peak area+uncut peak area).
  • XQ0001-5FAM GTATGTTGTGTGGAATTGTG 5'6-FAM;
  • XQ0002-5ROX GCTGCGCGTAACCACCACAC 5'ROX
  • the sequence of the amplification product is: GTATGTTGTGTGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGCTATGACCATGATTACGCCAAGCTGCCCTTCATTAGATCTGTGTGGCCAATTC TCCCCTACGTGCTGCTGAAG TTGCAAGGGCAGCTTCAATTCGCCCTATAGTGAGTCGTATTACAATTCACTGGCCGTCGTTTTACAACGTCGTGACTGGGAAAACC CTGGCGTTACCCCAACTTAATCGCCTTGCAGCACATCCCTTTCGCCAGCTGGCGTAATAGCGAAGAGGCCCGCACCGATCGCCCTTCCCAACAGTTGCGCAGCCTGAATGGCGAATGGACGCCCTGTAGCGGCGCATTAAGCGCGGCGGGTGTGGTGGTTACGCAGC; the part in italics is the PAM sequence, and the underlined area is the targeting region.
  • Cas-sf0005-5spacer1 GCCGUCAACGUUCAACGCUUGCUCGGUUCGCCGAGAC UCC CCUACGUGCUGCUGAAG (the underlined region is the target region)
  • reaction system 20 ⁇ L system, Tris-HAC 40mM, Mg(AC)2 30mM, BSA 120 ⁇ g/ml, DTT 12mM, pH7.0, Cas-sf0005 50nM, gRNA 100nM, double-stranded target nucleic acid 1 ⁇ L (PCR product). Incubate at 37°C for 5min, 15min, 30min, 60min, and at 85°C for 20min. Capillary electrophoresis detection (ABI 3730xl genetic analyzer) products in the FAM and ROX channels. The software Gene mapper 4.1 was used for data analysis, and the cutting efficiency of NTS/TS was calculated.
  • the cutting position of the Cas-sf0005 protein on the complementary strand and the non-complementary strand of the double-stranded target nucleic acid is determined by in vitro detection.
  • gRNA is used to guide the Cas-sf0005 protein to recognize and bind to the double-stranded target nucleic acid; the Cas-sf0005 protein stimulates the cleavage activity on the double-stranded target nucleic acid, thereby cutting the double-stranded target nucleic acid in the system.
  • the cleaved double-stranded target nucleic acid is filled in and A is added, and then the adapter with T is connected, and the ligated product is subjected to PCR enrichment and then sanger sequencing.
  • the target nucleic acid is selected as double-stranded DNA (plasmid).
  • the synthetic sequence: CATTAGATCTGTGTGGCCAATTC TCCCCTACGTGCTGCTGAAG TTGC is connected to the vector T-Vector-pEASY-Blunt Simple Cloning Vector.
  • the italic part is the PAM sequence, and the underlined area is the targeting region.
  • gRNA Cas-sf0005-5spacer1: GCCGUCAACGUUCAACGCUUGCUCGGUUCGCCGAGAC UCCCCUACGUGCUGCUGAAG (the underlined region is the targeting region).
  • reaction system 50 ⁇ L system, buffer: Tris-HAC 40mM, Mg(AC)2 30mM, BSA120 ⁇ g/ml, DTT 12mM, pH7.0, the final concentration of Cas was 100nM, the final concentration of gRNA was 250nM, double-stranded target nucleic acid 10ng/ ⁇ L (plasmid).
  • the cleavage site of Cas-sf0005 to the complementary strand of the target sequence is between the 21nt and 22nt of the 5' end of the PAM complementary sequence, and the cleavage site of Cas-sf0005 to the non-complementary strand of the target sequence is at the 3' end of the PAM sequence.
  • the gRNA guides the Cas-sf0005 protein to recognize and bind to the above-mentioned complementary strand, and the above-mentioned non-complementary strand is the DNA strand paired with the complementary strand.
  • Cas-sf9417 protein expression plasmid the nucleic acid sequence was optimized for human codons, and then gene synthesis was performed, and then connected into the Escherichia coli expression vector PeT28(a)+ vector.
  • the JM23119 promoter was added to the vector PeT28(a)+-Cas-sf9417 to initiate the transcription of Cas-sf9417CrRNA.
  • Formation vector PeT28(a)+-Cas-sf9417-JM23119-crRNA, crRNA sequence: GUGGGAACCCUUCCUGAUGGCUCGAUCCGUCGAGAC UCCCCUACGUGCUGC UGAAGUUGC is underlined as the target sequence; PAM library construction: synthetic sequence CGTGTTTCGTAAAGTCTGGAAACGCGGAAGCCCCCAGCGCTTCAGCGTT CNNNNNN TCCCCTACGTGCTGCTG AAGTTGC CCGCAA, N is random deoxynucleotide, underscore for the target sequence. After filling in with Klenow enzyme, it was connected into the pacyc184 vector. After transforming Escherichia coli, the plasmid was extracted to form a PAM library.
  • PAM library subtraction experiment Prepare competent state: BL21(DE3)-PeT28(a)+-Cas-sf9417-JM23119-crRNA.
  • PAM library plasmid transformation competence BL21(DE3)-PeT28(a)+-Cas-sf9417-JM23119-crRNA, spread on LB plates containing kanamycin and chloramphenicol, collect bacteria after overnight culture at 37°C For the body, adjust the concentration of the bacterial solution to OD600 0.6-0.8, add IPTG 0.2mM, and induce at 37°C for 4h.
  • FastPure EndoFree Plasmid Maxi Kit (vazyme) was used for plasmid extraction to obtain the reduced PAM library.
  • PAM-F GGTCTTCGGTTTCCGTGTT
  • PAM-R TGGCGTTGACTCTCAGTCAT.
  • the samples of the control group were obtained by PCR reaction using 30 ng/ ⁇ L plasmid (PAM library) as the template primer
  • the samples of the experimental group were obtained by PCR reaction using 30 ng/ ⁇ L plasmid (PAM library after subtraction) as the template.
  • the cis cleavage activity of the double-stranded DNA of Cas-sf9417 was detected in vitro.
  • the gRNA that can be paired with the target nucleic acid is used to guide the Cas-sf9417 protein to recognize and bind to the target nucleic acid, thereby cutting the target nucleic acid in the system, and the cleaved target nucleic acid is detected by agarose electrophoresis.
  • gRNA:Cas-sf9417-5spacer1 GUGGGAACCCUUCCUGAUGGCUCGAUCCGUCGAGAC UC CCCUACGUGCUGCUGAAG (underlined region is the target region)
  • the following reaction system was used: 20 ⁇ L system, buffer: Tris-HAC 40mM, Mg(AC)2 30mM, BSA 120 ⁇ g/ml, DTT 12mM, pH7.0, the final concentration of Cas-sf9417 was 100nM, the final concentration of gRNA was 200nM, double The final concentration of strand target nucleic acid was 5ng/ ⁇ L.
  • Incubate at 37°C for 1h and at 85°C for 20min.
  • the cleavage product was subjected to agarose electrophoresis to detect the cleavage ability of Cas-sf9417.
  • Cas-sf9417 protein, gRNA and target nucleic acid were added to the experimental group, and gRNA was not added to the control group (CK).
  • the gene editing activity of Cas-sf9417 protein was verified in animal cells, and the target was designed for the FUT8 gene of Chinese hamster ovary cells (CHO).
  • the vector pcDNA3.3 was transformed with EGFP fluorescent protein and PuroR resistance gene. Insert the SV40NLS-Cas-sf9417-NLS fusion protein through the restriction site BsmB1; insert the U6 promoter and gRNA sequence through the restriction site Mfe1.
  • the CMV promoter promotes the expression of the fusion protein SV40NLS-Cas-sf9417-NLS-GFP. Protein Cas-sf9417-NLS and protein GFP were connected with connecting peptide T2A.
  • the promoter EF-1 ⁇ drives the expression of the puromycin resistance gene.
  • Plating CHO cells are plated to a confluence of 70-80%, and the number of cells seeded in a 12-well plate is 8*10 ⁇ 4 cells/well.
  • Transfection plate for 12-24h for transfection, add 2ug plasmid to 100 ⁇ L opti-MEM and mix well; add 4 ⁇ L of the diluted plasmid EL Transfection Reagent (TRAN), incubate at room temperature for 15-20min. The incubated mixture was added to the culture medium on which the cells were layered for transfection.
  • TRAN plasmid EL Transfection Reagent
  • Genomic DNA extraction, PCR amplification near the edited region, sending to hiTOM for sequencing the cells were collected after trypsinization, and the genomic DNA was extracted with the Cell/Tissue Genomic DNA Extraction Kit (Bitec). Genomic DNA was amplified by primers NAFUT8-JC-Hitom-1F2: GGAGTGAGTACGGTGTGCAGTCCATGTCAGACGCACTG; PQ0106-FUT8-HiTom-R5: GAGTTGGATGCTGGATGGTACAGAACCACTTGTTGGTC to amplify the region near the target. PCR products were subjected to hiTOM sequencing (http://121.40.237.174/HiTOM/Sample_acceptance_sanyang.php).
  • Sequencing data analysis counting the sequence types and ratios within 20 bp upstream and downstream of the target-18 position, and obtaining the editing efficiency of the target position by the Cas-sf9417 protein.
  • CHO cell FUT8 gene target sequence gR16-FUT8: TTT AACAAAGAAGGGTCATCAGTGG CC , the italic part is the PAM sequence, and the underlined area is the targeting region.
  • the gRNA sequence is GUGGGAACCCUUCCUGAUGGCUCGAUCCGUCGAGAC AACAAAGAAGGGUCAUCAGUGG , and the underlined region is the targeting region.
  • the cutting efficiency of Cas-sf9417 on the complementary strand (TS) and the non-complementary strand (NTS) of the target nucleotide double-stranded DNA was tested in vitro.
  • 5'6-FAM marks the non-complementary strand (NTS)
  • 5'ROX marks the complementary strand (TS)
  • gRNA guides the Cas-sf9417 protein to recognize and bind to the complementary strand of the target nucleic acid, thereby cutting the target nucleic acid in the system, after cleavage
  • the target nucleic acid was detected by capillary electrophoresis (ABI 3730xl genetic analyzer).
  • the DNA fragments migrate from the cathode to the anode in the gel, and are arranged according to the length of the fragments.
  • the fluorescent dye When they migrate to the scanning window of the laser scanner at the anode, the fluorescent dye is excited and emits light of a certain wavelength, which is recorded according to the fluorescence intensity.
  • the electrophoretic traces of DNA fragments with fluorescent dyes are recorded according to the actual time when they pass through the laser scanning window, and each fragment is represented by a fluorescence absorption peak. The higher the peak value, the more fragments there are; the peak appearance time is directly related to the fragment size, the smaller the fragment, the earlier the peak appears.
  • the Cas-sf9417 uncut NTS fragment in the FAM fluorescence channel is 380nt, and the Cas-sf9417 cut NTS fragment is about 126nt.
  • XQ0001-5FAM GTATGTTGTGTGGAATTGTG 5'6-FAM;
  • XQ0002-5ROX GCTGCGCGTAACCACCACAC 5'ROX
  • the sequence of the amplification product is: GTATGTTGTGTGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGCTATGACCATGATTACGCCAAGCTGCCCTTCATTAGATCTGTGTGGCCAATTC TCCCCTACGTGCTGCTGAAG TTGCAAGGGCAGCTTCAATTCGCCCTATAGTGAGTCGTATTACAATTCACTGGCCGTCGTTTTACAACGTCGTGACTGGGAAAACC CTGGCGTTACCCCAACTTAATCGCCTTGCAGCACATCCCTTTCGCCAGCTGGCGTAATAGCGAAGAGGCCCGCACCGATCGCCCTTCCCAACAGTTGCGCAGCCTGAATGGCGAATGGACGCCCTGTAGCGGCGCATTAAGCGCGGCGGGTGTGGTGGTTACGCAGC; the part in italics is the PAM sequence, and the underlined area is the targeting region.
  • gRNA Cas-sf9417-5spacer1:GUGGGAACCCUUCCUGAUGGCUCGAUCCGUCGAGAC UCCCCUACGUGCUGCUGAAG (the underlined area is the target area)
  • reaction system 20 ⁇ L system, buffer: Tris-HAC 40mM, Mg(AC)2 30mM, BSA 120 ⁇ g/ml, DTT 12mM, pH7.0, Cas-sf9417 50nM, gRNA 100nM, double-stranded target nucleic acid 1 ⁇ L (PCR product). Incubate at 37°C for 5min, 15min, 30min, 60min, and at 85°C for 20min. Capillary electrophoresis detection (ABI 3730xl genetic analyzer) products in the FAM and ROX channels. The software Gene mapper 4.1 was used for data analysis, and the cutting efficiency of NTS and TS was calculated.
  • the cleaving position of the Cas-sf9417 protein on the complementary strand and the non-complementary strand of the double-stranded target nucleic acid is determined by in vitro detection.
  • gRNA is used to guide the Cas-sf9417 protein to recognize and bind to the double-stranded target nucleic acid; the Cas protein stimulates the cis cleavage activity on the double-stranded target nucleic acid, thereby cutting the double-stranded target nucleic acid in the system.
  • the cleaved double-stranded target nucleic acid is filled in and A is added, and then the adapter with T is connected, and the ligated product is subjected to PCR enrichment and then sanger sequencing.
  • the selected target nucleic acid is a double-stranded DNA (plasmid), sequence: CATTAGATCTGTGTGGCCAATTC TCCCCTACGTGCTGCTGAAG TTGC into the vector T-Vector-pEASY-Blunt Simple Cloning Vector, the italic part is the PAM sequence, and the underlined area is the targeting region.
  • plasmid double-stranded DNA
  • gRNA Cas-sf9417-5spacer1: GUGGGAACCCUUCCUGAUGGCUCGAUCCGUCGAGAC UCCCCUACGUGCUGCUGAAG (the underlined region is the targeting region).
  • reaction system 50 ⁇ L system, buffer: Tris-HAC 40mM, Mg(AC)2 30mM, BSA 120 ⁇ g/ml, DTT 12mM, pH7.0, Cas-sf9417 100nM, gRNA 250nM, double-stranded target nucleic acid 10ng/ ⁇ L (plasmid).
  • PCR products were subjected to Sanger sequencing. The results are shown in Figure 10, when the Cas-sf9417 protein cuts the target nucleic acid, the cutting position is in the middle of 14-16nt of NTS and the middle of 21-22nt of TS.
  • the cleavage site of Cas-sf9417 on the complementary strand of the target sequence is between the 21nt and 22nt of the 5' end of the PAM complementary sequence, and the cleavage site of Cas-sf9417 on the non-complementary strand of the target sequence is at the 3' end of the PAM sequence.
  • the gRNA guides the Cas-sf9417 protein to recognize and bind to the above-mentioned complementary strand, and the above-mentioned non-complementary strand is the DNA strand paired with the complementary strand.
  • Cas-sf8553 protein expression plasmid the nucleic acid sequence was optimized for human codons, and then gene synthesis was performed, and then connected into the E. coli expression vector PeT28(a)+ vector.
  • the JM23119 promoter was added to the vector PeT28(a)+-Cas-sf8553 to initiate the transcription of Cas-sf8553CrRNA.
  • PAM library subtraction experiment Prepare competent state: BL21(DE3)-PeT28(a)+-Cas-sf8553-JM23119-crRNA.
  • PAM library plasmid transformation competence BL21(DE3)-PeT28(a)+-Cas-sf8553-JM23119-crRNA, spread on LB plates containing kanamycin and chloramphenicol, collect bacteria after overnight culture at 37°C For the body, adjust the concentration of the bacterial solution to OD600 0.6-0.8, add IPTG 0.2mM, and induce at 37°C for 4h.
  • the large extraction kit is used for plasmid extraction to obtain the reduced PAM library.
  • PAM-F GGTCTTCGGTTTCCGTGTT
  • PAM-R TGGCGTTGACTCTCAGTCAT.
  • PAM library 30ng/ ⁇ L plasmid
  • PAM library after subtraction 30ng/ ⁇ L plasmid
  • the samples of the control group and the experimental group were sent to next-generation sequencing for data analysis.
  • This embodiment detects the cis cleavage activity of the double-stranded DNA of Cas-sf8553.
  • the gRNA that can be paired with the target nucleic acid is used to guide the Cas-sf8553 protein to recognize and bind to the target nucleic acid, thereby cutting the target nucleic acid in the system, and the cleaved target nucleic acid is detected by agarose electrophoresis.
  • Cas-sf8553-5spacer1 GUGAGGACGACAACCGAAGGCUCGGUGCGCCGAGAC UCCC CUACGUGCUGCUGAAG (the underlined region is the target region)
  • reaction system 20 ⁇ L system, buffer: Tris-HAC 40mM, Mg(AC)2 30mM, BSA 120 ⁇ g/ml, DTT 12mM, pH7.0, Cas-sf8553 100nM, gRNA 200nM, the final concentration of double-stranded target nucleic acid was 5ng/ ⁇ L.
  • buffer Tris-HAC 40mM, Mg(AC)2 30mM, BSA 120 ⁇ g/ml, DTT 12mM, pH7.0, Cas-sf8553 100nM, gRNA 200nM
  • the final concentration of double-stranded target nucleic acid was 5ng/ ⁇ L.
  • Incubate at 37°C for 1h and at 85°C for 20min.
  • the cleavage product was electrophoresed with 1% agarose to detect the cleavage ability of Cas-sf8553.
  • Cas-sf8553 protein, gRNA and target nucleic acid were added to the experimental group, and no g
  • the Cas-sf8553 protein was used to verify its gene editing activity in animal cells, and the target was designed for the FUT8 gene of Chinese hamster ovary cells (CHO).
  • the vector pcDNA3.3 was transformed with EGFP fluorescent protein and PuroR resistance gene. Insert the SV40NLS-Cas-sf8553-NLS fusion protein through the restriction site BsmB1; insert the U6 promoter and gRNA sequence through the restriction site Mfe1.
  • the CMV promoter promotes the expression of the fusion protein SV40NLS-Cas-sf8553-NLS-GFP. Protein Cas-sf8553-NLS and protein GFP were connected with connecting peptide T2A.
  • the promoter EF-1 ⁇ drives the expression of the puromycin resistance gene.
  • Plating CHO cells are plated to a confluence of 70-80%, and the number of cells seeded in a 12-well plate is 8*10 ⁇ 4 cells/well.
  • Transfection plate for 12-24h for transfection, add 2ug plasmid to 100 ⁇ L opti-MEM and mix well; add 4 ⁇ L of the diluted plasmid EL Transfection Reagent (TRAN), incubate at room temperature for 15-20min. The incubated mixture was added to the culture medium on which the cells were layered for transfection.
  • TRAN plasmid EL Transfection Reagent
  • Genomic DNA extraction, PCR amplification near the edited region, sending to hiTOM for sequencing the cells were collected after trypsinization, and the genomic DNA was extracted with the Cell/Tissue Genomic DNA Extraction Kit (Bitec). Genomic DNA was amplified by primers PQ0106-FUT8-HiTom-F1: GGAGTGAGTACGGTGTGCGAGTTCTGTTGCATGGTAGG; PQ0106-FUT8-HiTom-R1: GAGTTGGATGCTGGATGGGCCAAGCTTCTTGGTGGTTTC to amplify the region near the target. PCR products were subjected to hiTOM sequencing (http://121.40.237.174/HiTOM/Sample_acceptance_sanyang.php).
  • Sequencing data analysis counting the sequence types and ratios within 20 bp upstream and downstream of the target-18 position, and obtaining the editing efficiency of the Cas-sf0005 protein to the target position.
  • CHO cell FUT8 gene target sequence gR3-FUT8: TTC CAGCCAAGGTTGTGGACGGATC A , the italic part is the PAM sequence, and the underlined area is the targeting region.
  • the gRNA sequence is GUGAGGACGACAACCGAAGGCUCGGUGCGCCGAGACCAGCCAAGGUUGUGGACGGAUCA, and the underlined region is the targeting region.
  • the analysis results showed that the editing efficiency of Cas-sf8553 in the target gR3-FUT8 of CHO cells was 1.05%, and the editing type was InDel.
  • This embodiment detects the cutting efficiency of Cas-sf8553 on the target nucleotide (double-stranded DNA) complementary strand (TS) and non-complementary strand (NTS).
  • 5'6-FAM marks the non-complementary strand (NTS)
  • 5'ROX marks the complementary strand (TS)
  • gRNA guides the Cas-sf8553 protein to recognize and bind to the target nucleic acid, thereby cutting the target nucleic acid in the system, and the cleaved target nucleic acid Capillary electrophoresis detection (ABI 3730xl genetic analyzer).
  • the DNA fragments migrate from the cathode to the anode in the gel, and are arranged according to the length of the fragments.
  • the fluorescent dye When they migrate to the scanning window of the laser scanner at the anode, the fluorescent dye is excited and emits light of a certain wavelength, which is recorded according to the fluorescence intensity.
  • the electrophoretic traces of DNA fragments with fluorescent dyes are recorded according to the actual time when they pass through the laser scanning window, and each fragment is represented by a fluorescence absorption peak. The higher the peak value, the more fragments there are; the peak appearance time is directly related to the fragment size, the smaller the fragment, the earlier the peak appears.
  • the Cas-sf8553 uncut NTS fragment size is 380nt
  • the Cas-sf8553 cut NTS fragment is about 126nt.
  • the uncut TS fragment of Cas-sf8553 in the ROX fluorescence channel is 380 nt, and the TS fragment cut by Cas-sf8553 is about 254 nt.
  • Fragment cutting efficiency calculation formula: cutting efficiency cut peak area / (cut peak area + uncut peak area)
  • XQ0001-5FAM GTATGTTGTGTGGAATTGTG 5'6-FAM;
  • XQ0002-5ROX GCTGCGCGTAACCACCACAC 5'ROX
  • the sequence of the amplification product is: GTATGTTGTGTGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGCTATGACCATGATTACGCCAAGCTGCCCTTCATTAGATCTGTGTGGCCAATTC TCCCCTACGTGCTGCTGAAG TTGCAAGGGCAGCTTCAATTCGCCCTATAGTGAGTCGTATTACAATTCACTGGCCGTCGTTTTACAACGTCGTGACTGGGAAAACC CTGGCGTTACCCCAACTTAATCGCCTTGCAGCACATCCCTTTCGCCAGCTGGCGTAATAGCGAAGAGGCCCGCACCGATCGCCCTTCCCAACAGTTGCGCAGCCTGAATGGCGAATGGACGCCCTGTAGCGGCGCATTAAGCGCGGCGGGTGTGGTGGTTACGCAGC; the part in italics is the PAM sequence, and the underlined area is the targeting region.
  • gRNA Cas-sf8553-5spacer1: GUGAGGACGACAACCGAAGGCUCGGUGCGCCGAGAC UCCCCUACGUGCUGCUGAAG (the underlined region is the targeting region).
  • reaction system 20 ⁇ L system, buffer: Tris-HAC 40mM, Mg(AC)2 30mM, BSA 120 ⁇ g/ml, DTT 12mM, pH7.0, Cas-sf8553 50nM, gRNA 100nM, double-stranded target nucleic acid 1 ⁇ L (PCR product). Incubate at 37°C for 5min, 15min, 30min, 60min, and at 85°C for 20min. Capillary electrophoresis detection (ABI 3730xl genetic analyzer) products in the FAM and ROX channels. The software Gene mapper 4.1 was used for data analysis, and the cutting efficiency of NTS/TS was calculated.
  • the cleavage position of the complementary strand and the non-complementary strand of the double-stranded target nucleic acid of the Cas-sf8553 protein is determined by in vitro detection.
  • gRNA is used to guide the Cas-sf8553 protein to recognize and bind to the double-stranded target nucleic acid; the Cas-sf8553 protein stimulates the cis cleavage activity on the double-stranded target nucleic acid, thereby cutting the double-stranded target nucleic acid in the system.
  • the cleaved double-stranded target nucleic acid is filled in and A is added, and then the adapter with T is connected, and the ligated product is subjected to PCR enrichment and then sanger sequencing.
  • the selected target nucleic acid is a double-stranded DNA (plasmid), sequence: CATTAGATCTGTGTGGCCAATTC TCCCCTACGTGCTGCTGAAG TTGC into the vector T-Vector-pEASY-Blunt Simple Cloning Vector, the italic part is the PAM sequence, and the underlined area is the targeting region.
  • plasmid double-stranded DNA
  • gRNA Cas-sf8553-5spacer1:GUGAGGACGACAACCGAAGGCUCGGUGCGCCGAGAC UCCCCUACGUGCUGCUGAAG (underlined region is the target region)
  • reaction system 50 ⁇ L system, buffer: Tris-HAC 40mM, Mg(AC)2 30mM, BSA 120 ⁇ g/ml, DTT 12mM, pH7.0, Ca-sf8553 100nM, gRNA 250nM, double-stranded target nucleic acid 10ng/ ⁇ L (plasmid).
  • the cleavage site of Cas-sf8553 on the complementary strand of the target sequence is between the 21nt and 23nt of the 5' end of the PAM complementary sequence, and the cleavage site of Cas-sf8553 on the non-complementary strand of the target sequence is at the 3' end of the PAM sequence.
  • the gRNA guides the Cas-sf8553 protein to recognize and bind to the aforementioned complementary strand, and the aforementioned non-complementary strand is the DNA strand paired with the complementary strand.
  • the trans cleavage activity of Cas-sf1846 was verified by an in vitro detection test.
  • the gRNA that can be paired with the target nucleic acid is used to guide the Cas-sf1846 protein to recognize and bind to the target nucleic acid; then, the Cas-sf1846 protein stimulates the trans cleavage activity on any single-stranded nucleic acid, thereby cutting the single-stranded nucleic acid in the system detector; the two ends of the single-stranded nucleic acid detector are respectively provided with a fluorescent group and a quencher group, and if the single-stranded nucleic acid detector is cut, fluorescence will be excited; in other embodiments, the two ends of the single-stranded nucleic acid detector The end can also be provided with a label that can be detected by colloidal gold.
  • the selected target nucleic acid is single-stranded DNA, N-B-i3g1-ssDNA0, and its sequence is: CGACATTCCGAAGAACGCTGAAGCGCTGGGGGCAAATTGTGCAATTTGCGGC;
  • the gRNA sequence corresponding to Cas-sf1846 is: GUUGCAAGCCUUACUGAUUGCUCCUCACGAGGAGAC CCCCCAGCGCUUCAGCGUUC (the underlined region is the target region of gRNA);
  • the sequence of the single-stranded nucleic acid detector is FAM-TTATT-BHQ1;
  • the following reaction system was adopted: the final concentration of Cas-sf1846 was 50nM, the final concentration of gRNA was 50nM, the final concentration of target nucleic acid was 50nM, and the final concentration of single-stranded nucleic acid detector was 200nM. Incubate at 37°C and read FAM fluorescence/1min. In the control group, no target nucleic acid was added.

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Abstract

提供了一种新型的Cas酶，所述Cas酶为Cas-sf0005、Cas-sf9417、Cas-sf8553或Cas-sf1846，其与已报道的Cas酶的同源性较低，可以在细胞内和细胞外表现出核酸酶的活性，具有广泛的应用前景。

Description

新型的CRISPR酶和系统以及应用
本申请要求于2021年10月26日提交中国专利局、申请号为202111244315.7、发明名称为"新型的CRISPR酶和系统以及应用"的中国专利申请的优先权,其全部内容通过引用结合在本申请中。
技术领域
本发明涉及基因编辑领域，特别是规律成簇的间隔短回文重复(CRISPR)技术领域。具体而言，本发明涉及一种新型的CRISPR酶(或者，称之为CRISPR蛋白、Ca s效应蛋白、Cas酶或Cas蛋白)，包含此类蛋白的融合蛋白，以及编码它们的核酸分子。本发明还涉及用于核酸编辑(例如，基因或基因组编辑)的复合物和组合物，其包含本发明的Cas蛋白或融合蛋白，或编码它们的核酸分子。
背景技术
CRISPR/Cas技术是一种被广泛使用的基因编辑技术，它通过RNA引导对基因组上的靶序列进行特异性结合并切割DNA产生双链断裂，利用生物非同源末端连接或同源重组进行定点基因编辑。
CRISPR/Cas9系统是最常用的II型CRISPR系统，它识别3’-NGG的PAM基序，对靶标序列进行平末端切割。CRISPR/Cas Type V系统是一类新发现的CRISPR系统，它具有5’-TTN的基序，对靶标序列进行粘性末端切割，例如Cpf1,C2c1,CasX,CasY。然而目前存在的不同的CRISPR/Cas各有不同的优点和缺陷。例如Cas9,C2c1和CasX均需要两条RNA进行指导RNA，而Cpf1只需要一条指导RNA而且可以用来进行多重基因编辑。CasX具有980个氨基酸的大小，而常见的Cas9，C2c1，CasY和Cpf1通常大小在1300个氨基酸左右。此外，Cas9，Cpf1，CasX，CasY的PAM序列都比较复杂多样，而C2c1识别严谨的5’-TTN，因此它的靶标位点比其他系统容易被预测从而降低了潜在的脱靶效应。
总之，鉴于目前可获得的CRISPR/Cas系统都受限于一些缺陷，开发一种更稳健的、具有多方面良好性能的新型CRISPR/Cas系统对生物技术的发展具有重要意义。
发明内容
本申请的发明人经过大量实验和反复摸索，出人意料地发现了一种新型核酸内切酶(Cas酶)。基于这一发现，本发明人开发了新的CRISPR/Cas系统以及基于该系统的基因编辑方法和核酸检测方法。
Cas效应蛋白
一方面，本发明提供了一种Cas蛋白，所述Cas蛋白是CRISPR/Cas系统中的效应蛋白，在本发明中，将其称为Cas-sf0005(氨基酸序列如SEQ ID No.1所示)、Cas-sf9417(氨基酸序列如SEQ ID No.4所示)、Cas-sf8553(氨基酸序列如SEQ ID No.7所示)和Cas-sf1846(氨基酸序列如SEQ ID No.10所示)，进化树分析显示上述Cas蛋白属于
Figure PCTCN2022126671-appb-000001
类似的蛋白。
在一个实施方式中，所述Cas蛋白氨基酸序列与SEQ ID No.1或SEQ ID No.4或S EQ ID No.7或SEQ ID No.10相比具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、或至少99％的序列同一性，并且基本保留了其源自的序列的生物学功能。
在一个实施方式中，所述Cas蛋白氨基酸序列与SEQ ID No.1或SEQ ID No.4或S  EQ ID No.7或SEQ ID No.10相比，具有一个或多个氨基酸的置换、缺失或添加的序列；并且基本保留了其源自的序列的生物学功能；所述一个或多个氨基酸的置换、缺失或添加包括1个，2个，3个，4个，5个，6个，7个，8个，9个或10个氨基酸的置换、缺失或添加。
本领域技术人员清楚，可以改变蛋白质的结构而不对其活性和功能性产生不利影响，例如，可以在蛋白质氨基酸序列中引入一个或多个保守性氨基酸取代，而不会对蛋白质分子的活性和/或三维结构产生不利影响。本领域技术人员清楚保守性氨基酸取代的实例以及实施方式。具体的说，可以用与待取代位点属于相同组的另一氨基酸残基取代该氨基酸残基，即用非极性氨基酸残基取代另一非极性氨基酸残基，用极性不带电荷的氨基酸残基取代另一极性不带电荷的氨基酸残基，用碱性氨基酸残基取代另一碱性氨基酸残基，和用酸性氨基酸残基取代另一酸性氨基酸残基。这样的取代的氨基酸残基可以是也可以不是由遗传密码编码的。只要取代不导致蛋白质生物活性的失活，则一种氨基酸被属于同组的其他氨基酸替换的保守取代落在本发明的范围内。因此，本发明的蛋白可以在氨基酸序列中包含一个或多个保守性取代,这些保守性取代最好根据表1进行替换而产生。另外，本发明也涵盖还包含一个或多个其他非保守取代的蛋白，只要该非保守取代不显著影响本发明的蛋白质的所需功能和生物活性即可。
保守氨基酸置换可以在一个或多个预测的非必需氨基酸残基处进行。“非必需”氨基酸残基是可以发生改变(缺失、取代或置换)而不改变生物活性的氨基酸残基，而“必需”氨基酸残基是生物活性所需的。“保守氨基酸置换”是其中氨基酸残基被具有类似侧链的氨基酸残基替代的置换。氨基酸置换可以在上述Cas蛋白的非保守区域中进行。一般而言，此类置换不对保守的氨基酸残基，或者不对位于保守基序内的氨基酸残基进行，其中此类残基是蛋白质活性所需的。然而，本领域技术人员应当理解，功能变体可以具有较少的在保守区域中的保守或非保守改变。
表1
最初的残基 代表性的取代 优选的取代
Ala(A) Val；Leu；Ile Val
Arg(R) Lys；Gln；Asn Lys
Asn(N) Gln；His；Lys；Arg Gln
Asp(D) Glu Glu
Cys(C) Ser Ser
Gln(Q) Asn Asn
Glu(E) Asp Asp
Gly(G) Pro；Ala Ala
His(H) Asn；Gln；Lys；Arg Arg
Ile(I) Leu；Val；Met；Ala；Phe Leu
Leu(L) Ile；Val；Met；Ala；Phe Ile
Lys(K) Arg；Gln；Asn Arg
Met(M) Leu；Phe；Ile Leu
Phe(F) Leu；Val；Ile；Ala；Tyr Leu
Pro(P) Ala Ala
Ser(S) Thr Thr
Thr(T) Ser Ser
Trp(W) Tyr；Phe Tyr
Tyr(Y) Trp；Phe；Thr；Ser Phe
Val(V) Ile；Leu；Met；Phe；Ala Leu
本领域熟知，可以从蛋白质的N和/或C末端改变(置换、删除、截短或插入)一或多个氨基酸残基而仍保留其功能活性。因此，从Cas蛋白的N和/或C末端改变了一或多个氨基酸残基、同时保留了其所需功能活性的蛋白，也在本发明的范围内。这些改变可以包括通过现代分子方法例如PCR而引入的改变，所述方法包括借助于在PCR扩增中使用的寡核苷酸之中包含氨基酸编码序列而改变或延长蛋白质编码序列的PCR扩增。
应认识到，蛋白质可以以各种方式进行改变，包括氨基酸置换、删除、截短和插入，用于此类操作的方法是本领域通常已知的。例如，可以通过对DNA的突变来制备上述蛋白的氨基酸序列变体。还可以通过其他诱变形式和/或通过定向进化来完成，例如，使用已知的诱变、重组和/或改组(shuffling)方法，结合相关的筛选方法，来进行单个或多个氨基酸取代、缺失和/或插入。
领域技术人员能够理解，本发明Cas蛋白中的这些微小氨基酸变化可以出现(例如天然存在的突变)或者产生(例如使用r-DNA技术)而不损失蛋白质功能或活性。如果这些突变出现在蛋白的催化结构域、活性位点或其它功能结构域中，则多肽的性质可改变，但多肽可保持其活性。如果存在的突变不接近催化结构域、活性位点或其它功能结构域中，则可预期较小影响。
本领域技术人员可以根据本领域已知的方法，例如定位诱变或蛋白进化或生物信息系的分析，来鉴定本发明Cas蛋白的必需氨基酸。蛋白的催化结构域、活性位点或其它功能结构域也能够通过结构的物理分析而确定，如通过以下这些技术：如核磁共振、晶体学、电子衍射或光亲和标记，结合推定的关键位点氨基酸的突变来确定。
在一个实施方式中，所述Cas蛋白含有SEQ ID No.1或SEQ ID No.4或SEQ ID N o.7或SEQ ID No.10所示的氨基酸序列。
在一个实施方式中，所述Cas蛋白为SEQ ID No.1或SEQ ID No.4或SEQ ID No.7或SEQ ID No.10所示的氨基酸序列。
在一个实施方式中，所述Cas蛋白是与具有SEQ ID No.1或SEQ ID No.4或SEQ I D No.7或SEQ ID No.10所示的序列的蛋白质相同生物学功能的衍生化蛋白。
所述生物学功能包括但不限于，与指导RNA结合的活性、核酸内切酶活性、在指导RNA引导下与靶序列特定位点结合并切割的活性，包括但不限于Cis切割活性和Trans切割活性。
本发明还提供了一种融合蛋白，所述融合蛋白包括如上所述的Cas蛋白和其他的修饰部分。
在一个实施方式中，所述修饰部分选自另外的蛋白或多肽、可检测的标记或其任意组合。
在一个实施方式中，所述修饰部分选自表位标签、报告基因序列、核定位信号(NL S)序列、靶向部分、转录激活结构域(例如，VP64)、转录抑制结构域(例如，KRAB结构域或SID结构域)、核酸酶结构域(例如，Fok1)，以及具有选自下列的活性的结构域：核苷酸脱氨酶，甲基化酶活性,去甲基化酶,转录激活活性,转录抑制活性,转录释放因子活性,组蛋白修饰活性,核酸酶活性,单链RNA切割活性,双链RNA切割活性,单链DNA切割活性,双链DNA切割活性和核酸结合活性；以及其任意组合。所述NLS序列是本领域技术人员熟知的，其实例包括但不限于所述，SV40大T抗原，EGL-13，c-Myc以及TUS蛋白。
在一个实施方式中，所述NLS序列位于、靠近或接近本发明的Cas蛋白的末端(例 如，N端、C端或两端)。
所述表位标签(epitope tag)是本领域技术人员熟知的，包括但不限于His、V5、FLA G、HA、Myc、VSV-G、Trx等，并且本领域技术人员可以选择其他合适的表位标签(例如，纯化、检测或示踪)。
所述报告基因序列是本领域技术人员熟知的，其实例包括但不限于GST、HRP、CA T、GFP、HcRed、DsRed、CFP、YFP、BFP等。
在一个实施方式中，本发明的融合蛋白包含能够与DNA分子或细胞内分子结合的结构域，例如麦芽糖结合蛋白(MBP)、Lex A的DNA结合结构域(DBD)、GAL4的DBD等。
在一个实施方式中，本发明的融合蛋白包含可检测的标记，例如荧光染料，例如FIT C或DAPI。
在一个实施方式中，本发明的Cas蛋白任选地通过接头与所述修饰部分偶联、缀合或融合。
在一个实施方式中，所述修饰部分直接连接至本发明的Cas蛋白的N端或C端。
在一个实施方式中，所述修饰部分通过接头连接至本发明的Cas蛋白的N端或C端。这类接头是本领域熟知的，其实例包括但不限于包含一个或多个(例如，1个，2个，3个，4个或5个)氨基酸(如，Glu或Ser)或氨基酸衍生物(如，Ahx、β-Ala、G ABA或Ava)的接头，或PEG等。
本发明的Cas蛋白、蛋白衍生物或融合蛋白不受其产生方式的限定，例如，其可以通过基因工程方法(重组技术)产生，也可以通过化学合成方法产生。
Cas蛋白的核酸
另一方面，本发明提供了一种分离的多核苷酸，其包含：
(a)编码本发明的Cas蛋白或融合蛋白的多核苷酸序列；
(b)序列如SEQ ID No.2、3、5、6、8、9、11或12所示的多核苷酸；
(c)与SEQ ID No.2、3、5、6、8、9、11或12所示的序列相比具有一个或多个碱基的置换、缺失或添加(例如1个，2个，3个，4个，5个，6个，7个，8个，9个或10个碱基的置换、缺失或添加)的序列；
(d)核苷酸序列与SEQ ID No.2、3、5、6、8、9、11或12所示序列的同源性≥80％(较佳地≥90％，更佳地≥95％，最佳地≥98％)，且编码SEQ ID No.1或SEQ ID No.4或SE Q ID No.7或SEQ ID No.10所示多肽的多核苷酸；或者，
(e)与(a)-(d)任一所述的多核苷酸互补的多核苷酸。
在一个实施方式中，(a)-(e)任一项中所述的核苷酸序列经密码子优化用于在原核细胞中进行表达。在一个实施方式中，(a)-(e)任一项中所述的核苷酸序列经密码子优化用于在真核细胞中进行表达。
在一个实施方式中，所述的多核苷酸优选是单链的或双链的。
同向重复(Direct Repeat)序列
另一方面，本发明提供了一种与上述Cas蛋白形成复合物的工程化同向重复序列。
所述同向重复序列与能够和靶序列杂交的引导序列连接后构成指导RNA(guide RNA或gRNA)。
所述靶序列与gRNA的杂交，代表靶序列和gRNA的核酸序列至少70％，75％，80％，85％，90％，91％，92％，93％，94％，95％，96％，97％，98％，99％，或100％的同一性，从而可以杂交形成复合物；或者代表靶序列和gRNA的核酸序列至少有12个，15个，16个，17个，18个，19个，20个，21个，22个，或更多个碱基可以互补配对，形成复合物。
在一些实施例中，该同向重复序列与SEQ ID No.13-16任意一条序列具有至少90％的序列同一性。在一些实施例中，该同向重复序列与SEQ ID No.13-16任意一条所示的序列相比具有一个或多个碱基的置换、缺失或添加(例如1个，2个，3个，4个，5个，6个，7个，8个，9个或10个碱基的置换、缺失或添加)的序列。
在一些实施例中，同向重复序列如SEQ ID No.13-16任一所示。
指导RNA(gRNA)
另一方面，本发明提供了一种gRNA，所述gRNA包括第一区段和第二区段；所述第一区段又称为“骨架区”、“蛋白质结合区段”、“蛋白质结合序列”、或者“同向重复(Direct Repeat)序列”；所述第二区段又称为“靶向核酸的靶向序列”或者“靶向核酸的靶向区段”，或者“靶向靶序列的引导序列”。
所述gRNA的第一区段能够与本发明的Cas蛋白相互作用，从而使Cas蛋白和gRNA形成复合物。
在优选的实施方式中，所述第一区段为如上所述的同向重复序列。
本发明靶向核酸的靶向序列或靶向核酸的靶向区段包含与靶核酸中的序列互补的核苷酸序列。换言之，本发明靶向核酸的靶向序列或靶向核酸的靶向区段经过杂交(即，碱基配对)以序列特异性方式与靶核酸相互作用。因此，靶向核酸的靶向序列或靶向核酸的靶向区段可改变，或可被修饰以杂交靶核酸内的任何希望的序列。所述核酸选自DNA或R NA。
靶向核酸的靶向序列或靶向核酸的靶向区段与靶核酸的靶序列之间的互补百分比可为至少60％(例如，至少65％、至少70％、至少75％、至少80％、至少85％、至少90％、至少95％、至少97％、至少98％、至少99％或100％)。
本发明gRNA的“骨架区”、“蛋白质结合区段”、“蛋白质结合序列”、或者“同向重复序列”可以与CRISPR蛋白(或者，Cas蛋白)相互作用。本发明gRNA经过靶向核酸的靶向序列的作用将其相互作用的Cas蛋白引导至靶核酸内的特异性核苷酸序列。
优选的，所述指导RNA从5’至3’方向包含第一区段和第二区段。
本发明中，所述第二区段还可以理解为与靶序列杂交的引导序列。
本发明的gRNA能够与所述Cas蛋白形成复合物。
本发明的Cas-sf0005蛋白的gRNA包含与靶核酸杂交的引导序列，其中，所述靶核酸包括位于原型间隔区相邻基序(PAM)的3’端的序列；前述PAM序列为5’TBN-3’，其中，B＝T/C/G，N＝A/T/C/G。
本发明的Cas-sf9417蛋白的gRNA包含与靶核酸杂交的引导序列，其中，所述靶核酸包括位于原型间隔区相邻基序(PAM)的3’端的序列；前述PAM序列为5’-TTN-3’，其中，N＝A/T/C/G。
本发明的Cas-sf8553蛋白的gRNA包含与靶核酸杂交的引导序列，其中，所述靶核酸包括位于原型间隔区相邻基序(PAM)的3’端的序列；前述PAM序列为5’-TTN-3’，其中，N＝A/T/C/G。
载体
本发明还提供了一种载体，其包含如上述的Cas蛋白、分离的核酸分子或多核苷酸；优选的，其还包括与之可操作连接的调控元件。
在一个实施方式中，所述的调控元件选自下组中的一种或多种：增强子、转座子、启动子、终止子、前导序列、多腺苷酸序列、标记基因。
在一个实施方式中，所述的载体包括克隆载体、表达载体、穿梭载体、整合载体。
在一些实施方案中，所述系统中包括的载体是病毒载体(例如逆转录病毒载体，慢病毒载体，腺病毒载体，腺相关载体和单纯疱疹载体)，还可以是质粒、病毒、粘粒、噬菌 体等类型，它们是本领域技术人员所熟知的。
CRISPR系统
本发明提供了一种工程化的非天然存在的载体系统，或者是CRISPR-Cas系统，该系统包括Cas蛋白或编码所述Cas蛋白的核酸序列以及编码一种或多种指导RNA的核酸。
在一种实施方式中，所述编码所述Cas蛋白的核酸序列和编码一种或多种指导RNA的核酸是人工合成的。
在一种实施方式中，所述编码所述Cas蛋白的核酸序列和编码一种或多种指导RNA的核酸并不共同天然存在。
该一种或多种指导RNA在细胞中靶向一个或多个靶序列。所述一个或多个靶序列与编码一种或多种基因产物的DNA分子的基因组座位杂交，并且引导该Cas蛋白到达所述一种或多种基因产物的DNA分子的基因组座位部位，Cas蛋白到达靶序列位置后对靶序列进行修饰、编辑或切割，由此该一种或多种基因产物的表达被改变或修饰。
本发明的细胞包括动物、植物或微生物中的一种或多种。
在一些实施例中，该Cas蛋白是密码子优化的，用于在细胞中进行表达。
在一些实施例中，该Cas蛋白切割在该靶序列位置处的一条或两条链。
在一些实施例中，该Cas蛋白在gRNA的介导下切割靶核酸的互补链和/或非互补链。
优选的，该Cas蛋白同时切割靶核酸的互补链和非互补链。
本发明中，所述gRNA引导Cas蛋白识别并结合在所述互补链上，所述非互补链是与互补链配对的核酸链。PAM序列位于非互补链上，所述互补链含有与上述PAM序列配对的PAM互补序列。
在一个实施方式中，Cas-sf0005对靶序列的互补链的切割位点在PAM互补序列5’端第21nt和22nt之间，Cas-sf0005对靶序列的非互补链的切割位点在PAM序列3’端第15nt和16nt之间，gRNA引导Cas-sf0005蛋白识别并结合上述互补链上，上述非互补链是与互补链配对的DNA链。
在一个实施方式中，Cas-sf9417对靶序列的互补链的切割位点在PAM互补序列5’端第21nt和22nt之间，Cas-sf9417对靶序列的非互补链的切割位点在PAM序列3’端第14nt和16nt之间，gRNA引导Cas-sf9417蛋白识别并结合上述互补链上，上述非互补链是与互补链配对的DNA链。
在一个实施方式中，Cas-sf8553对靶序列的互补链的切割位点在PAM互补序列5’端第21nt和23nt之间，Cas-sf8553对靶序列的非互补链的切割位点在PAM序列3’端第14nt和15nt之间，gRNA引导Cas-sf8553蛋白识别并结合上述互补链上，上述非互补链是与互补链配对的DNA链。
本发明还提供了一种工程化的非天然存在的载体系统，该载体系统可以包括一种或多种载体，该一种或多种载体包括：
a)第一调控元件，该第一调控元件可操作地与gRNA连接，
b)第二调控元件，该第二调控元件可操作地与所述Cas蛋白连接；
其中组分(a)和(b)位于该系统的相同或不同载体上。
所述第一和第二调控元件包括启动子(例如，组成型启动子或诱导型启动子)、增强子(例如35S promoter或35S enhanced promoter)、内部核糖体进入位点(IRES)、和其他表达控制元件(例如转录终止信号，如多聚腺苷酸化信号和多聚U序列)。
在一些实施方案中，所述系统中的载体是病毒载体(例如逆转录病毒载体，慢病毒载体，腺病毒载体，腺相关载体和单纯疱疹载体)，还可以是质粒、病毒、粘粒、噬菌体等类型，它们是本领域技术人员所熟知的。
在一些实施例中，本文提供的系统处于递送系统中。在一些实施方案中，递送系统是纳米颗粒，脂质体，外体，微泡和基因枪。
在一个实施方式中，所述靶序列是来自原核细胞或真核细胞的DNA或RNA序列。在一个实施方式中，所述靶序列是非天然存在的DNA或RNA序列。
在一个实施方式中，所述靶序列存在于细胞内。在一个实施方式中，所述靶序列存在于细胞核内或细胞质(例如，细胞器)内。在一个实施方式中，所述细胞是真核细胞。在其他实施方式中，所述细胞是原核细胞。
在一个实施方式中，所述Cas蛋白连接有一个或多个NLS序列。在一个实施方式中，所述融合蛋白包含一个或多个NLS序列。在一个实施方式中，所述NLS序列连接至所述蛋白的N端或C端。在一个实施方式中，所述NLS序列融合至所述蛋白的N端或C端。
另一方面，本发明涉及一种工程化的CRISPR系统，所述系统包含上述Cas蛋白以及一种或多种指导RNA，其中，所述指导RNA包括同向重复序列和能够与靶核酸杂交的间隔序列，所述Cas蛋白能够结合所述指导RNA并靶向与间隔序列互补的靶核酸序列。
在一个实施方式中，所述Cas酶为Cas-sf0005，所述靶核酸为DNA(优选，双链DN A)，所述靶核酸位于原间隔序列临近基序(PAM)的3’端，并且所述PAM具有5’TBN-3’所示的序列，其中，B＝T/C/G，N＝A/T/C/G。
在一个实施方式中，所述Cas酶为Cas-sf9417或Cas-sf8553，所述靶核酸为DNA(优选，双链DNA)时，所述靶核酸位于原间隔序列临近基序(PAM)的3’端，并且所述P AM为5’-TTN-3’，其中，N＝A/T/C/G。
蛋白-核酸复合物/组合物
另一方面，本发明提供了一种复合物或者组合物，其包含：
(i)蛋白组分，其选自：上述Cas蛋白、衍生化蛋白或融合蛋白，及其任意组合；和
(ii)核酸组分，其包含(a)能够与靶序列杂交的引导序列；以及(b)能够与本发明的Cas蛋白结合的同向重复序列。
所述蛋白组分与核酸组分相互结合形成复合物。
在一个实施方式中，所述核酸组分是CRISPR-Cas系统中的指导RNA。
在一个实施方式中，所述复合物或组合物是非天然存在的或经修饰的。在一个实施方式中，所述复合物或组合物中的至少一个组分是非天然存在的或经修饰的。在一个实施方式中，所述第一组分是非天然存在的或经修饰的；和/或，所述第二组分是非天然存在的或经修饰的。
活化的CRISPR复合物
另一方面，本发明还提供了一种活化的CRISPR复合物，所述活化的CRISPR复合物包含：(1)蛋白组分，其选自：本发明的Cas蛋白、衍生化蛋白或融合蛋白，及其任意组合；(2)gRNA，其包含(a)能够与靶序列杂交的引导序列；以及(b)能够与本发明的Cas蛋白结合的同向重复序列；以及(3)结合在gRNA上的靶序列。优选的，所述结合为通过gRNA上的靶向核酸的靶向序列与靶核酸进行的结合。
本文所用术语“活化的CRISPR复合物”，“活化复合物”或“三元复合物”是指CRISPR系统中Cas蛋白、gRNA与靶核酸结合或修饰后的复合物。
本发明的Cas蛋白和gRNA可以形成二元复合物，该二元复合物在与核酸底物结合时被活化，形成活化的CRISPR复合物该核酸底物与gRNA中的间隔序列(或者称之为，与靶核酸杂交的引导序列)互补。在一些实施方案中，gRNA的间隔序列与靶底物完全匹配。在其它实施方案中，gRNA的间隔序列与靶底物的部分(连续或不连续)匹配。
在优选的实施方式中，所述活化的CRISPR复合物可以表现出侧枝核酸酶切活性，所 述侧枝核酸酶切活性是指活化的CRISPR复合物表现的对单链核酸的非特异切割活性或乱切活性，在本领域又称之为trans切割活性。
递送及递送组合物
本发明的Cas蛋白、gRNA、融合蛋白、核酸分子、载体、系统、复合物和组合物，可以通过本领域已知的任何方法进行递送。此类方法包括但不限于，电穿孔、脂转染、核转染、显微注射、声孔效应、基因枪、磷酸钙介导的转染、阳离子转染、脂质体转染、树枝状转染、热激转染、核转染、磁转染、脂转染、穿刺转染、光学转染、试剂增强性核酸摄取、以及经由脂质体、免疫脂质体、病毒颗粒、人工病毒体等的递送。
因此，在另一个方面，本发明提供了一种递送组合物，其包含递送载体，以及选自下列的一种或任意几种：本发明的Cas蛋白、融合蛋白、核酸分子、载体、系统、复合物和组合物。
在一个实施方式中，所述递送载体是粒子。
在一个实施方式中，所述递送载体选自脂质颗粒、糖颗粒、金属颗粒、蛋白颗粒、脂质体、外泌体、微泡、基因枪或病毒载体(例如，复制缺陷型逆转录病毒、慢病毒、腺病毒或腺相关病毒)。
宿主细胞
本发明还涉及一种体外的、离体的或体内的细胞或细胞系或它们的子代，所述细胞或细胞系或它们的子代包含：本发明所述的Cas蛋白、融合蛋白、核酸分子、蛋白-核酸复合物、活化的CRISPR复合物、载体、本发明递送组合物。
在某些实施方案中，所述细胞是原核细胞。
在某些实施方案中，所述细胞是真核细胞。在某些实施方案中，所述细胞是哺乳动物细胞。在某些实施方案中，所述细胞是人类细胞。某些实施方案中，所述细胞是非人哺乳动物细胞，例如非人灵长类动物、牛、羊、猪、犬、猴、兔、啮齿类(如大鼠或小鼠)的细胞。在某些实施方案中，所述细胞是非哺乳动物真核细胞，例如家禽鸟类(如鸡)、鱼类或甲壳动物(如蛤蜊、虾)的细胞。在某些实施方案中，所述细胞是植物细胞，例如单子叶植物或双子叶植物具有的细胞或栽培植物或粮食作物如木薯、玉米、高粱、大豆、小麦、燕麦或水稻具有的细胞，例如藻类、树或生产植物、果实或蔬菜(例如，树类如柑橘树、坚果树；茄属植物、棉花、烟草、番茄、葡萄、咖啡、可可等)。
在某些实施方案中，所述细胞是干细胞或干细胞系。
在某些情况下，本发明的宿主细胞包含基因或基因组的修饰，该修饰是在其野生型中不存在的修饰。
基因编辑方法和应用
本发明的Cas蛋白、核酸、上述组合物、上述CIRSPR/Cas系统、上述载体系统、上述递送组合物或上述活化的CRISPR复合物或者上述宿主细胞可用于以下任一或任意几个用途：靶向和/或编辑靶核酸；切割双链DNA、单链DNA或单链RNA；非特异性切割和/或降解侧枝核酸；非特异性切割单链核酸；核酸检测；检测目标样品中的核酸；特异性地编辑双链核酸；碱基编辑双链核酸；碱基编辑单链核酸。在其他的实施方式中，还可以用于制备用于上述任一或任意几个用途的试剂或试剂盒。
本发明还提供了上述Cas蛋白、核酸、上述组合物、上述CIRSPR/Cas系统、上述载体系统、上述递送组合物或上述活化的CRISPR复合物在基因编辑、基因靶向或基因切割中的应用；或者，在制备用于基因编辑、基因靶向或基因切割的试剂或试剂盒中的用途。
在一个实施方式中，所述基因编辑、基因靶向或基因切割为在细胞内和/或细胞外进行基因编辑、基因靶向或基因切割。
本发明还提供了一种编辑靶核酸、靶向靶核酸或切割靶核酸的方法，所述方法包括将 靶核酸与上述Cas蛋白、核酸、上述组合物、上述CIRSPR/Cas系统、上述载体系统、上述递送组合物或上述活化的CRISPR复合物进行接触。在一个实施方式中，所述方法为在细胞内或细胞外编辑靶核酸、靶向靶核酸或切割靶核酸。
所述基因编辑或编辑靶核酸包括修饰基因、敲除基因、改变基因产物的表达、修复突变、和/或插入多核苷酸、基因突变。
所述编辑可以在原核细胞和/或真核细胞中进行编辑。
另一方面，本发明还提供了上述Cas蛋白、核酸、上述组合物、上述CIRSPR/Cas系统、上述载体系统、上述递送组合物或上述活化的CRISPR复合物在核酸检测中的应用，或在制备用于核酸检测的试剂或试剂盒中的用途。
另一方面，本发明还提供了一种切割单链核酸的方法，所述方法包括，使核酸群体与上述Cas蛋白和gRNA接触，其中所述核酸群体包含靶核酸和多个非靶单链核酸，所述Cas蛋白切割所述多个非靶单链核酸。
所述gRNA能够结合所述Cas蛋白。
所述gRNA能够靶向所述靶核酸。
所述接触可以是在体外、离体或体内的细胞内部。
优选的，所述切割单链核酸为非特异性的切割。
另一方面，本发明还提供了上述Cas蛋白、核酸、上述组合物、上述CIRSPR/Cas系统、上述载体系统、上述递送组合物或上述活化的CRISPR复合物在非特异性的切割单链核酸中的应用，或在制备用于非特异性的切割单链核酸的试剂或试剂盒中的用途。
另一方面，本发明还提供了一种用于基因编辑、基因靶向或基因切割的试剂盒，所述试剂盒包括上述Cas蛋白、gRNA、核酸、上述组合物、上述CIRSPR/Cas系统、上述载体系统、上述递送组合物、上述活化的CRISPR复合物或上述宿主细胞。
另一方面，本发明还提供了一种用于检测样品中的靶核酸的试剂盒，所述试剂盒包含：(a)Cas蛋白，或编码所述Cas蛋白的核酸；(b)指导RNA，或编码所述指导RNA的核酸，或包含所述指导RNA的前体RNA，或编码所述前体RNA的核酸；和(c)为单链的且不与所述指导RNA杂交的单链核酸检测器。
本领域知晓，前体RNA可被切割或加工成为上述成熟的指导RNA。
另一方面，发明提供了上述Cas蛋白、核酸、上述组合物、上述CIRSPR/Cas系统、上述载体系统、上述递送组合物、上述活化的CRISPR复合物或上述宿主细胞在制备制剂或试剂盒中的用途，所述制剂或试剂盒用于：
(i)基因或基因组编辑；
(ii)靶核酸检测和/或诊断；
(iii)编辑靶基因座中的靶序列来修饰生物或非人类生物；
(iv)疾病的治疗；
(iv)靶向靶基因。
优选的，上述基因或基因组编辑为在细胞内或细胞外进行基因或基因组编辑。
优选的，所述靶核酸检测和/或诊断为在体外进行靶核酸检测和/或诊断。
优选的，所述疾病的治疗为治疗由靶基因座中的靶序列的缺陷引起的病症。
另一个方面，本发明提供了一种检测样品中靶核酸的方法，所述方法包括将样品与所述Cas蛋白、gRNA(指导RNA)和单链核酸检测器接触，所述gRNA包括与所述Cas蛋白结合的区域和与靶核酸杂交的指导序列；检测由所述Cas蛋白切割单链核酸检测器产生的可检测信号，从而检测靶核酸；所述单链核酸检测器不与所述gRNA杂交。
特异性修饰靶核酸的方法
另一方面，本发明还提供了一种特异性修饰靶核酸的方法，方法包括：使靶核酸与上 述Cas蛋白、核酸、上述组合物、上述CIRSPR/Cas系统、上述载体系统、上述递送组合物或上述活化的CRISPR复合物接触。
该特异性修饰可以发生在体内或者体外。
该特异性修饰可以发生在细胞内或者细胞外。
在一些情况下，细胞选自原核细胞或真核细胞，例如，动物细胞、植物细胞或微生物细胞。
在一个实施方式中，所述修饰是指所述靶序列的断裂，如，DNA的单链/双链断裂，或者RNA的单链断裂。
在一些情况下，所述方法还包括使靶核酸与供体多核苷酸接触，其中将供体多核苷酸、供体多核苷酸的部分、供体多核苷酸的拷贝或供体多核苷酸的拷贝的部分整合到靶核酸中。
在一个实施方式中，所述修饰还包括将编辑模板(例如外源核酸)插入所述断裂中。
在一个实施方式中，所述方法还包括：将编辑模板与所述靶核酸接触，或者递送至包含所述靶核酸的细胞中。在此实施方式中，所述方法通过与外源模板多核苷酸同源重组修复所述断裂的靶基因；在一些实施方式中，所述修复导致一种突变，包括所述靶基因的一个或多个核苷酸的插入、缺失、或取代，在其他的实施方式中，所述突变导致在从包含该靶序列的基因表达的蛋白质中的一个或多个氨基酸改变。
检测(非特异切割)
另一方面，本发明提供了一种检测样品中靶核酸的方法，所述方法包括将样品与上述Cas蛋白、核酸、上述组合物、上述CIRSPR/Cas系统、上述载体系统、上述递送组合物或上述活化的CRISPR复合物和单链核酸检测器接触；检测由所述Cas蛋白切割单链核酸检测器产生的可检测信号，从而检测靶核酸。
本发明中，所述靶核酸包括核糖核苷酸或脱氧核糖核苷酸；包括单链核酸、双链核酸，例如单链DNA、双链DNA、单链RNA、双链RNA。
在一个实施方式中，所述靶核酸来源于病毒、细菌、微生物、土壤、水源、人体、动物、植物等样品。优选的，所述靶核酸为PCR、NASBA、RPA、SDA、LAMP、HAD、NEAR、MDA、RCA、LCR、RAM等方法富集或扩增的产物。
在一个实施方式中，所述靶核酸为病毒核酸、细菌核酸、与疾病相关的特异核酸，如特定的突变位点或SNP位点或与对照有差异的核酸；优选地，所述病毒为植物病毒或动物病毒，例如，乳头瘤病毒，肝DNA病毒，疱疹病毒，腺病毒，痘病毒，细小病毒，冠状病毒；优选地，所述病毒为冠状病毒，优选地，SARS、SARS-CoV2(COVID-19)、HCoV-229E、HCoV-OC43、HCoV-NL63、HCoV-HKU1、Mers-Cov。
本发明中，所述gRNA与靶核酸上的靶序列至少有50％的匹配度，优选至少60％，优选至少70％，优选至少80％，优选至少90％。
在一个实施方式中，当所述的靶序列含有一个或多个特征位点(如特定的突变位点或SNP)时，所述的特征位点与gRNA完全匹配。
在一个实施方式中，所述检测方法中可以包含一种或多种导向序列互不相同的gRNA，其靶向不同的靶序列。
本发明中，所述单链核酸检测器包括但不限于单链DNA、单链RNA、DNA-RNA杂交体、核酸类似物、碱基修饰物、以及含有无碱基间隔物的单链核酸检测器等；“核酸类似物”包括但不限于：锁核酸、桥核酸、吗啉核酸、乙二醇核酸、己糖醇核酸、苏糖核酸、阿拉伯糖核酸、2’氧甲基RNA、2’甲氧基乙酰基RNA、2’氟RNA、2’氨基RNA、4’硫RNA及其组合，包括任选的核糖核苷酸或脱氧核糖核苷酸残基。
本发明中，所述可检测信号通过以下方式实现：基于视觉的检测，基于传感器的检 测，颜色检测，基于荧光信号的检测，基于金纳米颗粒的检测，荧光偏振，胶体相变/分散，电化学检测和基于半导体的检测。
本发明中，优选的，所述单链核酸检测器的两端分别设置荧光基团和淬灭基团，当所述单链核酸检测器被切割后，可以表现出可检测的荧光信号。所述荧光基团选自FAM、FITC、VIC、JOE、TET、CY3、CY5、ROX、Texas Red或LC RED460中的一种或任意几种；所述淬灭基团选自BHQ1、BHQ2、BHQ3、Dabcy1或Tamra中的一种或任意几种。
在其他的实施方式中，所述单链核酸检测器的5’端和3’端分别设置不同的标记分子，通过胶体金检测的方式，检测所述单链核酸检测器被Cas蛋白切割前和被Cas蛋白切割后的胶体金测试结果；所述单链核酸检测器被Cas蛋白切割前和被Cas蛋白切割后在胶体金的检测线和质控线上将表现出不同的显色结果。
在一些实施方案中，检测靶核酸的方法还可以包括将可检测信号的电平与参考信号电平进行比较，以及基于可检测信号的电平确定样品中靶核酸的量。
在一些实施方案中，检测靶核酸的方法还可以包括在不同的通道上使用RNA报告核酸和DNA报告核酸(例如，荧光颜色)，并通过测量RNA和DNA报告分子的信号电平，以及通过测量RNA和DNA报告分子中靶核酸的量来确定可检测信号的电平，基于组合(例如，使用最小或乘积)可检测信号的电平来采样。
在一个实施方式中，所述靶基因存在于细胞内。
在一个实施方式中，所述细胞是原核细胞。
在一个实施方式中，所述细胞是真核细胞。
在一个实施方式中，所述细胞是动物细胞。
在一个实施方式中，所述细胞是人类细胞。
在一个实施方式中，所述细胞是植物细胞，例如栽培植物(如木薯、玉米、高粱、小麦或水稻)、藻类、树或蔬菜具有的细胞。
在一个实施方式中，所述靶基因存在于体外的核酸分子(例如，质粒)中。
在一个实施方式中，所述靶基因存在于质粒中。
术语定义
在本发明中，除非另有说明，否则本文中使用的科学和技术名词具有本领域技术人员所通常理解的含义。并且，本文中所用的分子遗传学、核酸化学、化学、分子生物学、生物化学、细胞培养、微生物学、细胞生物学、基因组学和重组DNA等操作步骤均为相应领域内广泛使用的常规步骤。同时，为了更好地理解本发明，下面提供相关术语的定义和解释。
Cas蛋白
在本发明中，Cas蛋白、Cas酶、Cas效应蛋白可以互换使用；本发明人首次发现并鉴定了一种Cas效应蛋白，其具有选自下列的氨基酸序列：
(i)SEQ ID No.1或SEQ ID No.4或SEQ ID No.7或SEQ ID No.10；
(ii)与SEQ ID No.1或SEQ ID No.4或SEQ ID No.7或SEQ ID No.10所示的序列相比具有一个或多个氨基酸的置换、缺失或添加(例如1个，2个，3个，4个，5个，6个，7个，8个，9个或10个氨基酸的置换、缺失或添加)的序列；或
(iii)与SEQ ID No.1或SEQ ID No.4或SEQ ID No.7或SEQ ID No.10所示的序列具有至少20％、至少30％、至少40％、至少50％、至少60％、至少70％、至少80％、至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、或至少99％的序列同一性的序列。
本文中的核酸切割或切割核酸包括:由本文所述Cas酶产生的靶核酸中的DNA或RN  A断裂(Cis切割)、DNA或RNA在侧枝核酸底物(单链核酸底物)中的断裂(即非特异性或非靶向性，Trans切割)。在一些实施方式中，所述切割是双链DNA断裂。在一些实施方案中，切割是单链DNA断裂或单链RNA断裂。
CRISPR系统
如本文中所使用的，术语“规律成簇的间隔短回文重复(CRISPR)-CRISPR-相关(Cas)(CRISPR-Cas)系统”或“CRISPR系统”可互换地使用并且具有本领域技术人员通常理解的含义，其通常包含与CRISPR相关(“Cas”)基因的表达有关的转录产物或其他元件，或者能够指导所述Cas基因活性的转录产物或其他元件。
CRISPR/Cas复合物
如本文中所使用的，术语“CRISPR/Cas复合物”是指，指导RNA(guide RNA)或成熟c rRNA与Cas蛋白结合所形成的复合体，其包含杂交到靶序列的引导序列上并且与Cas蛋白结合的同向重复序列，该复合体能够识别并切割能与该指导RNA或成熟crRNA杂交的多核苷酸。
指导RNA(guide RNA，gRNA)
如本文中所使用的，术语“指导RNA(guide RNA，gRNA)”、“成熟crRNA”、“指导序列”可互换地使用并且具有本领域技术人员通常理解的含义。一般而言，指导RNA可以包含同向重复序列(direct repeat)和引导序列，或者基本上由或由同向重复序列和引导序列组成。
在某些情况下，指导序列是与靶序列具有足够互补性从而与所述靶序列杂交并引导C RISPR/Cas复合物与所述靶序列的特异性结合的任何多核苷酸序列。在一个实施方式中，当最佳比对时，指导序列与其相应靶序列之间的互补程度为至少50％、至少60％、至少70％、至少80％、至少90％、至少95％、或至少99％。确定最佳比对在本领域的普通技术人员的能力范围内。例如，存在公开和可商购的比对算法和程序，诸如但不限于Clustal W、matlab中的史密斯-沃特曼算法(Smith-Waterman)、Bowtie、Geneious、Biopython以及SeqMan。
靶序列
“靶序列”是指被gRNA中的引导序列所靶向的多核苷酸，例如与该引导序列具有互补性的序列，其中靶序列与引导序列之间的杂交将促进CRISPR/Cas复合物(包括Cas蛋白和gRNA)的形成。完全互补性不是必需的，只要存在足够互补性以引起杂交并且促进一种CRISPR/Cas复合物的形成即可。
靶序列可以包含任何多核苷酸，如DNA或RNA。在某些情况下，所述靶序列位于细胞内或细胞外。在某些情况下，所述靶序列位于细胞的细胞核或细胞质中。在某些情况下，该靶序列可位于真核细胞的一个细胞器例如线粒体或叶绿体内。可被用于重组到包含该靶序列的靶基因座中的序列或模板被称为“编辑模板”或“编辑多核苷酸”或“编辑序列”。在一个实施方式中，所述编辑模板为外源核酸。在一个实施方式中，该重组是同源重组。
在本发明中，“靶序列”或“靶多核苷酸”或“靶核酸”可以是对细胞(例如，真核细胞)而言任何内源或外源的多核苷酸。例如，该靶多核苷酸可以是一种存在于真核细胞的细胞核中的多核苷酸。该靶多核苷酸可以是一个编码基因产物(例如，蛋白质)的序列或一个非编码序列(例如，调节多核苷酸或无用DNA)。在某些情况下，该靶序列应该与原间隔序列临近基序(PAM)相关。
单链核酸检测器
本发明所述的单链核酸检测器是指含有2-200个核苷酸的序列，优选，具有2-150个核苷酸，优选，3-100个核苷酸，优选，3-30个核苷酸，优选，4-20个核苷酸，更优选，5-15个核苷酸。优选为单链DNA分子、单链RNA分子或单链DNA-RNA杂交体。
所述的单链核酸检测器两端包括不同的报告基团或标记分子，当其处于初始状态(即未被切割状态时)不呈现报告信号，当该单链核酸检测器被切割后，呈现出可检测的信号，即切割后与切割前表现出可检测的区别。
在一个实施方式中，所述的报告基团或标记分子包括荧光基团和淬灭基团，所述荧光基团选自FAM、FITC、VIC、JOE、TET、CY3、CY5、ROX、Texas Red或LC RED460中的一种或任意几种；所述淬灭基团选自BHQ1、BHQ2、BHQ3、Dabcy1或Tamra中的一种或任意几种。
在一个实施方式中，所述的单链核酸检测器具有连接至5’端第一分子(如FAM或FI TC)和连接至3’端的第二分子(如生物素)。所述的含有单链核酸检测器的反应体系与流动条配合用以检测靶核酸(优选，胶体金检测方式)。所述的流动条被设计为具有两条捕获线，在样品接触端(胶体金)设有结合第一分子的抗体(即第一分子抗体)，在第一线(control line)处含有结合第一分子抗体的抗体，在第二线(test line)处含有与第二分子结合的第二分子的抗体(即第二分子抗体，如亲和素)。当反应沿着条带流动时，第一分子抗体与第一分子结合携带切割或未切割的寡核苷酸至捕获线，切割的报告子将在第一个捕获线处结合第一分子抗体的抗体，而未切割的报告子将在第二捕获线处结合第二分子抗体。报告基团在各条线的结合将导致强读出/信号(例如颜色)。随着更多的报告子被切割，更多的信号将在第一捕获线处累积，并且在第二线处将出现更少的信号。在某些方面，本发明涉及如本文所述的流动条用于检测核酸的用途。在某些方面，本发明涉及用本文定义的流动条检测核酸的方法，例如(侧)流测试或(侧)流免疫色谱测定。在某些方面，所述单链核酸检测器中的分子可相互替换，或改变分子的位置，只要其报告原理与本发明相同或相近，所改进的方式也均包含在本发明中。
本发明所述的检测方法，可用于待检测靶核酸的定量检测。所述的定量检测指标可以根据报告基团的信号强弱进行定量，如根据荧光基团的发光强度，或根据显色条带的宽度等。
野生型
如本文中所使用的，术语“野生型”具有本领域技术人员通常理解的含义，其表示生物、菌株、基因的典型形式或者当它在自然界存在时区别于突变体或变体形式的特征，其可从自然中的来源分离并且没有被人为有意地修饰。
衍生化
如本文中所使用的，术语“衍生化”是指，对氨基酸、多肽或蛋白的化学修饰，其中一个或多个取代基已与所述氨基酸、多肽或蛋白共价连接。取代基也可称为侧链。
衍生化的蛋白是该蛋白的衍生物，通常，蛋白的衍生化不会不利影响该蛋白的期望活性(例如，与指导RNA结合的活性、核酸内切酶活性、在指导RNA引导下与靶序列特定位点结合并切割的活性)，也就是说蛋白的衍生物与蛋白有相同的活性。
衍生化蛋白
又称“蛋白衍生物”，是指蛋白的经修饰形式，例如其中所述蛋白的一个或多个氨基酸可以被缺失、插入、修饰和/或取代。
非天然存在的
如本文中所使用的，术语“非天然存在的”或“工程化的”可互换地使用并且表示人工的参与。当这些术语用于描述核酸分子或多肽时，其表示该核酸分子或多肽至少基本上从它们在自然界中或如发现于自然界中的与其结合的至少另一种组分游离出来。
直系同源物(orthologue,ortholog)
如本文中所使用的，术语“直系同源物(orthologue,ortholog)”具有本领域技术人员通常理解的含义。作为进一步指导，如本文中所述的蛋白质的“直系同源物”是指属于不同 物种的蛋白质，该蛋白质执行与作为其直系同源物的蛋白相同或相似的功能。
同一性
如本文中所使用的，术语“同一性”用于指两个多肽之间或两个核酸之间序列的匹配情况。当两个进行比较的序列中的某个位置都被相同的碱基或氨基酸单体亚单元占据时(例如，两个DNA分子的每一个中的某个位置都被腺嘌呤占据，或两个多肽的每一个中的某个位置都被赖氨酸占据)，那么各分子在该位置上是同一的。两个序列之间的“百分数同一性”是由这两个序列共有的匹配位置数目除以进行比较的位置数目×100的函数。例如，如果两个序列的10个位置中有6个匹配，那么这两个序列具有60％的同一性。例如，DNA序列CTGACT和CAGGTT共有50％的同一性(总共6个位置中有3个位置匹配)。通常，在将两个序列比对以产生最大同一性时进行比较。这样的比对可通过使用，例如，可通过计算机程序例如Align程序(DNAstar,Inc.)方便地进行的Needleman等人(1970)J.Mol.Biol.48：443-453的方法来实现。还可使用已整合入ALIGN程序(版本2.0)的E.Meyers和W.Miller(Comput.Appl Biosci.，4:11-17(1988))的算法，使用PAM120权重残基表(weight residue table)、12的缺口长度罚分和4的缺口罚分来测定两个氨基酸序列之间的百分数同一性。此外，可使用已整合入GCG软件包(可在www.gcg.com上获得)的GAP程序中的Needleman和Wunsch(J MoI Biol.48:444-453(1970))算法，使用Blossum 62矩阵或PAM250矩阵以及16、14、12、10、8、6或4的缺口权重(gap weight)和1、2、3、4、5或6的长度权重来测定两个氨基酸序列之间的百分数同一性。
载体
术语“载体”是指一种核酸分子，它能够运送与其连接的另一种核酸分子。载体包括但不限于，单链、双链、或部分双链的核酸分子；包括一个或多个自由端、无自由端(例如环状的)的核酸分子；包括DNA、RNA、或两者的核酸分子；以及本领域已知的其他多种多样的多核苷酸。载体可以通过转化，转导或者转染导入宿主细胞，使其携带的遗传物质元件在宿主细胞中获得表达。一种载体可以被引入到宿主细胞中而由此产生转录物、蛋白质、或肽，包括由如本文所述的蛋白、融合蛋白、分离的核酸分子等(例如，CRISPR转录物，如核酸转录物、蛋白质、或酶)。一种载体可以含有多种控制表达的元件，包括但不限于，启动子序列、转录起始序列、增强子序列、选择元件及报告基因。另外，载体还可含有复制起始位点。
一种类型的载体是“质粒”，其是指其中可以例如通过标准分子克隆技术插入另外的DNA片段的环状双链DNA环。
另一种类型的载体是病毒载体，其中病毒衍生的DNA或RNA序列存在于用于包装病毒(例如，逆转录病毒、复制缺陷型逆转录病毒、腺病毒、复制缺陷型腺病毒、以及腺相关病毒)的载体中。病毒载体还包含由用于转染到一种宿主细胞中的病毒携带的多核苷酸。某些载体(例如，具有细菌复制起点的细菌载体和附加型哺乳动物载体)能够在它们被导入的宿主细胞中自主复制。
其他载体(例如，非附加型哺乳动物载体)在引入宿主细胞后整合到该宿主细胞的基因组中，并且由此与该宿主基因组一起复制。而且，某些载体能够指导它们可操作连接的基因的表达。这样的载体在此被称为“表达载体”。
宿主细胞
如本文中所使用的，术语“宿主细胞”是指，可用于导入载体的细胞，其包括但不限于，如大肠杆菌或枯草菌等的原核细胞，如微生物细胞、真菌细胞、动物细胞和植物细胞的真核细胞。
本领域技术人员将理解，表达载体的设计可取决于诸如待转化的宿主细胞的选择、所希望的表达水平等因素。
调控元件
如本文中所使用的，术语“调控元件”旨在包括启动子、增强子、内部核糖体进入位点(IRES)、和其他表达控制元件(例如转录终止信号，如多聚腺苷酸化信号和多聚U序列)，其详细描述可参考戈德尔(Goeddel)，《基因表达技术：酶学方法》(GENE EXPRESSION TECHNOLOGY:METHODS IN ENZYMOLOGY)185，学术出版社(Academic Press)，圣地亚哥(San Diego)，加利福尼亚州(1990)。在某些情况下，调控元件包括指导一个核苷酸序列在许多类型的宿主细胞中的组成型表达的那些序列以及指导该核苷酸序列只在某些宿主细胞中表达的那些序列(例如，组织特异型调节序列)。组织特异型启动子可主要指导在感兴趣的期望组织中的表达，所述组织例如肌肉、神经元、骨、皮肤、血液、特定的器官(例如肝脏、胰腺)、或特殊的细胞类型(例如淋巴细胞)。在某些情况下，调控元件还可以时序依赖性方式(如以细胞周期依赖性或发育阶段依赖性方式)指导表达，该方式可以是或者可以不是组织或细胞类型特异性的。在某些情况下，术语“调控元件”涵盖的是增强子元件，如WPRE；CMV增强子；在HTLV-I的LTR中的R-U5’片段((Mol.Cell.Biol.，第8(1)卷，第466-472页，1988)；SV40增强子；以及在兔β-珠蛋白的外显子2与3之间的内含子序列(Proc.Natl.Acad.Sci.USA.，第78(3)卷，第1527-31页，1981)。
启动子
如本文中所使用的，术语“启动子”具有本领域技术人员公知的含义，其是指一段位于基因的上游能启动下游基因表达的非编码核苷酸序列。组成型(constitutive)启动子是这样的核苷酸序列：当其与编码或者限定基因产物的多核苷酸可操作地相连时，在细胞的大多数或者所有生理条件下，其导致细胞中基因产物的产生。诱导型启动子是这样的核苷酸序列，当可操作地与编码或者限定基因产物的多核苷酸相连时，基本上只有当对应于所述启动子的诱导物在细胞中存在时，其导致所述基因产物在细胞内产生。组织特异性启动子是这样的核苷酸序列：当可操作地与编码或者限定基因产物的多核苷酸相连时，基本上只有当细胞是该启动子对应的组织类型的细胞时，其才导致在细胞中产生基因产物。
NLS
“核定位信号”或“核定位序列”(NLS)是对蛋白质“加标签”以通过核转运导入细胞核的氨基酸序列，即，具有NLS的蛋白质被转运至细胞核。典型地，NLS包含暴露在蛋白质表面的带正电荷的Lys或Arg残基。示例性核定位序列包括但不限于来自以下的NLS：SV40大T抗原，EGL-13，c-Myc以及TUS蛋白。在一些实施例中，该NLS包含PKKKRKV序列。在一些实施例中，该NLS包含AVKRPAATKKAGQAKKKKLD序列。在一些实施例中，该NLS包含PAAKRVKLD序列。在一些实施例中，该NLS包含MSRRRKANPTKLSENAKKLAKEVEN序列。在一些实施例中，该NLS包含KLKIKRPVK序列。其他核定位序列包括但不限于hnRNP A1的酸性M9结构域、酵母转录抑制子Matα2中的序列KIPIK和PY-NLS。
可操作地连接
如本文中所使用的，术语“可操作地连接”旨在表示感兴趣的核苷酸序列以一种允许该核苷酸序列的表达的方式被连接至该一种或多种调控元件(例如，处于一种体外转录/翻译系统中或当该载体被引入到宿主细胞中时，处于该宿主细胞中)。
互补性
如本文中所使用的，术语“互补性”是指核酸与另一个核酸序列借助于传统的沃森-克里克或其他非传统类型形成一个或多个氢键的能力。互补百分比表示一个核酸分子中可与一个第二核酸序列形成氢键(例如，沃森-克里克碱基配对)的残基的百分比(例如，10个之中有5、6、7、8、9、10个即为50％、60％、70％、80％、90％、和100％互补)。“完全互补”表示一个核酸序列的所有连续残基与一个第二核酸序列中的相同数目的连续残基形 成氢键。如本文使用的“基本上互补”是指在一个具有8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、30、35、40、45、50个或更多个核苷酸的区域上至少为60％、65％、70％、75％、80％、85％、90％、95％、97％、98％、99％、或100％的互补程度，或者是指在严格条件下杂交的两个核酸。
严格条件
如本文中所使用的，对于杂交的“严格条件”是指与靶序列具有互补性的一个核酸主要地与该靶序列杂交并且基本上不杂交到非靶序列上的条件。严格条件通常是序列依赖性的，并且取决于许多因素而变化。一般而言，该序列越长，则该序列特异性地杂交到其靶序列上的温度就越高。
杂交
术语“杂交”或“互补的”或“基本上互补的”是指核酸(例如RNA、DNA)包含使其能够非共价结合的核苷酸序列，即以序列特异性，反平行的方式(即核酸特异性结合互补核酸)与另一核酸形成碱基对和/或G/U碱基对，“退火”或“杂交”。
杂交需要两个核酸含有互补序列，尽管碱基之间可能存在错配。两个核酸之间杂交的合适条件取决于核酸的长度和互补程度，这是本领域公知的变量。典型地，可杂交核酸的长度为8个核苷酸或更多(例如，10个核苷酸或更多，12个核苷酸或更多，15个核苷酸或更多，20个核苷酸或更多，22个核苷酸或更多，25个核苷酸或更多，或30个核苷酸或更多)。
应当理解，多核苷酸的序列不需要与其靶核酸的序列100％互补以特异性杂交。多核苷酸可包含60％或更高，65％或更高，70％或更高，75％或更高，80％或更高，85％或更高，90％或更高，95％或更高，98％或更高，99％或更高，99.5％或更高，或与其杂交的靶核酸序列中的靶区域的序列互补性为100％。
靶序列与gRNA的杂交代表靶序列和gRNA的核酸序列至少60％、70％、75％、80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％的可以杂交，形成复合物；或者代表靶序列和gRNA的核酸序列至少有12个、15个、16个、17个、18个、19个、20个、21个、22个或更多个碱基可以互补配对，杂交形成复合物。
表达
如本文中所使用的，术语“表达”是指，藉此从DNA模板转录成多核苷酸(如转录成mRNA或其他RNA转录物)的过程和/或转录的mRNA随后藉此翻译成肽、多肽或蛋白质的过程。转录物和编码的多肽可以总称为“基因产物”。如果多核苷酸来源于基因组DNA，表达可以包括真核细胞中mRNA的剪接。
接头
如本文中所使用的，术语“接头”是指，由多个氨基酸残基通过肽键连接形成的线性多肽。本发明的接头可以为人工合成的氨基酸序列，或天然存在的多肽序列，例如具有铰链区功能的多肽。此类接头多肽是本领域众所周知的(参见例如，Holliger,P.等人(1993)Proc.Natl.Acad.Sci.USA 90:6444-6448；Poljak,R.J.等人(1994)Structure 2:1121-1123)。
治疗
如本文中所使用的，术语“治疗”是指，治疗或治愈病症，延缓病症的症状的发作，和/或延缓病症的发展。
受试者
如本文中所使用的，术语“受试者”包括但不限于各种动物、植物和微生物。
动物
例如哺乳动物，例如牛科动物、马科动物、羊科动物、猪科动物、犬科动物、猫科动物、兔科动物、啮齿类动物(例如，小鼠或大鼠)、非人灵长类动物(例如，猕猴或食蟹猴)或人。在某些实施方式中，所述受试者(例如人)患有病症(例如，疾病相关基因缺陷所导致的病症)。
植物
术语“植物”应理解为能够进行光合作用的任何分化的多细胞生物，在包括处于任何成熟或发育阶段的作物植物，特别是单子叶或双子叶植物，蔬菜作物，包括洋蓟、球茎甘蓝、芝麻菜、韭葱、芦笋、莴苣(例如，结球莴苣、叶莴苣、长叶莴苣)、小白菜(bok choy)、黄肉芋、瓜类(例如，甜瓜、西瓜、克伦肖瓜(crenshaw)、白兰瓜、罗马甜瓜)、油菜作物(例如，球芽甘蓝、卷心菜、花椰菜、西兰花、羽衣甘蓝、无头甘蓝、大白菜、小白菜)、刺菜蓟、胡萝卜、洋白菜(napa)、秋葵、洋葱、芹菜、欧芹、鹰嘴豆、欧洲防风草、菊苣、胡椒、马铃薯、葫芦(例如，西葫芦、黄瓜、小西葫芦、倭瓜、南瓜)、萝卜、干球洋葱、芜菁甘蓝、紫茄子(也称为茄子)、婆罗门参、苣菜、青葱、苦苣、大蒜、菠菜、绿洋葱、倭瓜、绿叶菜类(greens)、甜菜(糖甜菜和饲料甜菜)、甘薯、唐莴苣、山葵、西红柿、芜菁、以及香辛料；水果和/或蔓生作物，如苹果、杏、樱桃、油桃、桃、梨、李子、西梅、樱桃、榅桲、杏仁、栗子、榛子、山核桃、开心果、胡桃、柑橘、蓝莓、博伊增莓(boysenberry)、小红莓、穗醋栗、罗甘莓、树莓、草莓、黑莓、葡萄、鳄梨、香蕉、猕猴桃、柿子、石榴、菠萝、热带水果、梨果、瓜、芒果、木瓜、以及荔枝；大田作物，如三叶草、苜蓿、月见草、白芒花、玉米/玉蜀黍(饲料玉米、甜玉米、爆米花)、啤酒花、荷荷芭、花生、稻、红花、小粒谷类作物(大麦、燕麦、黑麦、小麦等)、高粱、烟草、木棉、豆科植物(豆类、小扁豆、豌豆、大豆)、含油植物(油菜、芥菜、罂粟、橄榄、向日葵、椰子、蓖麻油植物、可可豆、落花生)、拟南芥属、纤维植物(棉花、亚麻、大麻、黄麻)、樟科(肉桂、莰酮)、或一种植物如咖啡、甘蔗、茶、以及天然橡胶植物；和/或花坛植物，如开花植物、仙人掌、肉质植物和/或观赏植物，以及树如森林(阔叶树和常绿树，如针叶树)、果树、观赏树、以及结坚果的树(nut-bearing tree)、以及灌木和其他苗木。
发明的有益效果
本发明发现了一种新型的Cas酶，Blast结果显示，本申请的Cas酶与已经报道的Cas酶的一致性较低，其可以在体内和体外表现出核酸酶的活性，具有广泛的应用前景。
下面将结合附图和实施例对本发明的实施方案进行详细描述，但是本领域技术人员将理解，下列附图和实施例仅用于说明本发明，而不是对本发明的范围的限定。根据附图和优选实施方案的下列详细描述，本发明的各种目的和有利方面对于本领域技术人员来说将变得显然。
附图说明
图1.Cas-sf0005的PAM结构。
图2.Cas-sf0005对双链核酸的切割结果。
图3.Cas-sf0005对真核细胞编辑后的靶基因测序结果。
图4.Cas-sf0005对互补链和非互补链的切割效率。
图5.Cas-sf0005切割靶核酸的位置。
图6.Cas-sf9417的PAM结构。
图7.Cas-sf9417对双链核酸的切割结果。
图8.Cas-sf9417对真核细胞编辑后的靶基因测序结果。
图9.Cas-sf9417对互补链和非互补链的切割效率。
图10.Cas-sf9417切割靶核酸的位置。
图11.Cas-sf8553的PAM结构。
图12.Cas-sf8553对双链核酸的切割结果。
图13.Cas-sf8553对互补链和非互补链的切割效率。
图14.Cas-sf8553切割靶核酸的位置。
图15.Cas-sf1846用于核酸检测的荧光结果图。
序列信息
Figure PCTCN2022126671-appb-000002
具体实施方式
以下实施例仅用于描述本发明，而非限定本发明。除非特别指明，否则基本上按照本领域内熟知的以及在各种参考文献中描述的常规方法进行实施例中描述的实验和方法。例如，本发明中所使用的免疫学、生物化学、化学、分子生物学、微生物学、细胞生物学、基因组学和重组DNA等常规技术，可参见萨姆布鲁克(Sambrook)、弗里奇(Fritsch)和马尼亚蒂斯(Maniatis)，《分子克隆：实验室手册》(MOLECULAR CL ONING:A LABORATORY MANUAL)，第2次编辑(1989)；《当代分子生物学实验手册》(CURRENT PROTOCOLS IN MOLECULAR BIOLOGY)(F.M.奥苏贝尔(F.M.Ausubel)等人编辑，(1987))；《酶学方法》(METHODS IN ENZYMOLOGY)系列(学术出版公司)：《PCR 2：实用方法》(PCR 2:A PRACTICAL APPROACH)(M.J.麦克弗森(M.J.MacPherson)、B.D.黑姆斯(B.D.Hames)和G.R.泰勒(G.R.Taylor)编辑(1995))、哈洛(Harlow)和拉内(Lane)编辑(1988)《抗体：实验室手册》(ANTIBODIES,A LABORATO RY MANUAL)，以及《动物细胞培养》(ANIMAL CELL CULTURE)(R.I.弗雷谢尼(R.I.Freshney)编辑(1987))。
另外，实施例中未注明具体条件者，按照常规条件或制造商建议的条件进行。所用试剂或仪器未注明生产厂商者，均为可以通过市购获得的常规产品。本领域技术人员知 晓，实施例以举例方式描述本发明，且不意欲限制本发明所要求保护的范围。本文中提及的全部公开案和其他参考资料以其全文通过引用合并入本文。
实施例1.Cas蛋白的获得
发明人对未培养物的宏基因组进行分析，通过对去冗余、蛋白质聚类分析，鉴定得到了4个新的Cas酶。Blast结果显示，Cas蛋白与已报道的Cas蛋白的序列一致性较低，进化树分析显示其属于
Figure PCTCN2022126671-appb-000003
类似的蛋白，本发明中将其命名为Cas-sf0005、Cas-sf9417、Cas-sf8553和Cas-sf1846。
上述蛋白的氨基酸序列、编码核酸序列以及人源密码子优化后的核酸序列如下所示：
Figure PCTCN2022126671-appb-000004
对上述蛋白所对应的gRNA的同向重复序列进行分析，结果显示：
Cas-sf0005蛋白对应的gRNA的同向重复序列为CCGUCAACGUUCAACGCUUGCUCGGUUCGCCGAGAC(SEQ ID No.13)或者是CCGUCAACGUUCAACGCUUGCUCGGUACGCCGAGAC；
Cas-sf9417蛋白对应的gRNA的同向重复序列为GUGGGAACCCUUCCUGAUGGCUCGAUCCGUCGAGAC(SEQ ID No.14)；
Cas-sf8553蛋白对应的gRNA的同向重复序列为GUGAGGACGACAACCGAAGGCUCGGUGCGCCGAGAC(SEQ ID No.15)；
Cas-sf1846蛋白所对应的gRNA的同向重复序列为GUUGCAAGCCUUACUGAUUGCUCCUCACGAGGAGAC(SEQ ID No.16)。
实施例2.Cas-sf0005蛋白PAM鉴定
构建PAM文库，合成序列:CGTGTTTCGTAAAGTCTGGAAACGCGGAAGCCCCCAGCGCTTCAGCGTTCNNNNNN TCCCCTACGTGCTGCTGAAGTTGCCCGCAA，N为随机脱氧核苷酸，下划线为靶点序列。经Klenow酶补平后连入pacyc184载体。转化大肠杆菌后提取质粒形成PAM文库。gRNA Cas-sf0005-5'spacer1 20bp序列为：CCGUCAACGUUCAACGCUUGCUCGGUUCGCCGAGAC UCCCCUACGUGCUGCUGAAG(下划线区域为靶向区)；
引物序列：TK-117：CGGCATTCCTGCTGAACCGCTCTTCCGATCT；
TK-111：GATCGGAAGAGCGGTTCAGCAGGAATGCCG；
PAM-after-F：ACTCAGGGGTCTTCGGTTTCCGTGTT；
S6-PAM-after：ACTCAGCTGAACCGCTCTTCCG；
获取Cas-sf0005蛋白偏好PAM库：50nM Cas-sf0005蛋白，50nM gRNA在缓冲液Tris-HAC 40mM、Mg(AC)2 30mM、BSA 120μg/ml、DTT 12mM、pH7.0中，25℃孵育10min。添加PAM文库质粒(10ng/μL)，37℃孵育1h；85℃孵育20min。在上述体系中添加2.5U DreamTaq DNA Polymerase(5U/μL)(Thermo Fisher Scientific)和4μL 2.5mM d NTP Mix(全式金)，72℃孵育30min，进行切割末端的补平及3’端加A。对上述产物经试剂盒(Omega Gel Extraction Kit D2500)进行产物纯化。引物TK-117和TK-111退火后与上述产物经T4连接酶连接。得到的连接产物用引物PAM-after-F和S6-PAM-after进行PCR反应得到Cas-sf0005蛋白偏好PAM库，PCR产物进行二代测序，经分析得到PAM序列，使用Weblogo作图，结果如图1所示，Cas-sf0005识别的PAM序列为5’TBN-3’，其中，B＝T/C/G，N＝A/T/C/G。
实施例3.Cas-sf0005蛋白在进行双链核酸编辑的应用
本实施例通过体外检测测试Cas-sf0005蛋白对双链DNA的切割活性。本实施例中利用可以与靶核酸配对的gRNA引导Cas-蛋白识别并结合在双链靶核酸上；随后，Cas蛋白激发对双链靶核酸的切割活性，从而切割体系里的双链靶核酸。切割后的双链靶核酸进行琼脂糖凝胶电泳检测。
本实施例中选择靶核酸为双链DNA，其序列为：GAACGCTGAAGCGCTGGGGGCATTA TCCCCTACGTGCTGCTGAAGTTGC连入载体T-Vector-pEASY-Blunt Simple Cloning Vector；斜体部分TTA为PAM序列，下划线区域为靶向区。gRNA-Cas-sf0005-5'spacer1-20bp：CCGUCAACGUUCAACGCUUGCUCGGUUCGCCGAGAC UCCCCUACGUGCUGCU GAAG(下划线区域为靶向区)；同时将上述片段PCR扩增后作为靶核酸检测；采用如下反应体系：20μL体系，Tris-HAC 40mM、Mg(AC)2 30mM、BSA 120μg/ml、DTT 12mM、pH7.0，Cas-sf0005 50nM，gRNA 500nM，双链靶核酸终浓度为5ng/μL。Cas蛋白、gRNA，25℃孵育10min；加入双链靶核酸，37℃孵育1h。1.0％琼脂糖凝胶电泳检测。实验组添加靶核酸和gRNA，对照组不添加gRNA。
如图2A所示(从左至右依次为泳道1、泳道2和泳道3)，泳道1为实验组，泳道2为对照组、泳道3为Trans2K plus DNA marker。与不添加gRNA的对照相比，实验组的Cas-sf0005在含有靶核酸(质粒)和gRNA时能够有效切割体系里的双链核酸。图2B所示，泳道1为Trans2K plus DNA marker，泳道2-5为不同PAM的实验组、泳道3为对照组。与不添加gRNA的对照相比，实验组的Cas-sf0005在含有TTN PAM的靶核酸(PCR产物)和gRNA时能够有效切割体系里的双链核酸。实验结果表明Cas-sf0005可以用于双链靶核酸的切割和编辑。
实施例4.Cas-sf0005蛋白在动物细胞中的编辑效率
采用Cas-sf0005蛋白在动物细胞中验证其基因编辑的活性，针对中国仓鼠卵巢细胞(CHO)FUT8基因设计靶点。载体pcDNA3.3经改造后带有EGFP荧光蛋白及PuroR抗性基因。经酶切位点BsmB1插入SV40NLS-Cas-sf0005融合蛋白；经酶切位点Mfe1插入U6启动子及gRNA序列。CMV启动子启动融合蛋白SV40NLS-Cas-sf0005-NLS-GFP表达。蛋白Cas-sf0005-NLS与蛋白GFP用连接肽T2A进行连接。启动子EF-1α启动嘌呤霉素抗性基因表达。
铺板：CHO细胞融合度至70-80％进行铺板，12孔板中接种细胞数为8*10^4细胞/孔。
转染：铺板6-8h进行转染，125μl opti-MEM中加入3.25μl Lipo3000混匀；125μl opti-MEM中加入3ug质粒、10μl P3000,混匀。稀释好的Lipo3000与稀释后的质粒混合均匀，室温孵育5min。孵育好的混合液加入铺有细胞的培养基中进行转染。
加嘌呤霉素筛选：转染24h加嘌呤霉素，终浓度10ng/ml。嘌呤霉素处理24h更换成 正常培养基继续培养24h。
提DNA、PCR扩增编辑区附近、送hiTOM测序：细胞经胰酶消化处理后进行收集，经细胞/组织基因组DNA提取试剂盒(百泰克)进行基因组DNA提取。基因组DNA经引物NAFUT8-JC-HITOM-1F1：GGAGTGAGTACGGTGTGCTCCTCCTTACTTACCCTTGG；NAFUT8-JC-HITOM-1R1：GAGTTGGATGCTGGATGGTTGTTCTTTGGTGGGACTATG扩增靶点附近区域。PCR产物进行hiTOM测序(http://121.40.237.174/HiTOM/Sample_acceptance_sanyang.php)。
测序数据分析，统计靶点位置上游15nt、下游10nt范围内的序列种类及比例，得到Cas-sf0005蛋白对靶点位置的编辑效率。
CHO细胞FUT8基因靶点序列：gR1-FUT8：TTT GACAAACTGGGATACCCACCAC A，斜体部分为PAM序列，下划线区域为靶向区。gRNA序列为GCCGUCAACGUUCAACGCUUGCUCGGUUCGCCGAGAC GACAAACUGGGAUACCCACCACA，下划线区域为靶向区。
分析结果显示，Cas-sf0005在CHO细胞的靶点gR1-FUT8中的编辑效率达到22.49％，编辑类型均InDel，经编辑后的靶核酸的部分测序结果如图3所示。
实施例5.Cas-sf0005蛋白的切割特性-NTS/TS切割效率
本实施例通过体外检测Cas-sf0005对靶核苷酸(双链DNA)互补链(TS)、非互补链(NTS)的切割效率。5'6-FAM标记非互补链(NTS)、5'ROX标记互补链(TS)，gRNA引导Cas-sf0005蛋白识别并结合在靶核酸互补链(TS)上，从而切割体系里的靶核酸的互补链(TS)与非互补链(NTS)，切割后的靶核酸进行毛细管电泳检测(ABI 3730xl遗传分析仪)。DNA片段在凝胶中从阴极向阳极迁移，按片段长度大小排列，当迁移到阳极端的激光扫描仪的扫描窗口，荧光染料受到激发，发出一定波长的光，按荧光强度记录下来，每一个带荧光染料的DNA片段电泳轨迹按各自通过激光扫描窗口的实际时间被记录下来，以荧光吸收峰表示每一个片段。峰值越高，表示该片段量越多；峰出现的时间与片段大小有直接关系，片段越小，峰越早出现。FAM荧光通道中Cas-sf0005未切割NTS片段大小为380nt，Cas-sf0005切割NTS后片段约126nt。ROX荧光通道中Cas-sf0005未切割TS片段大小为380nt，Cas-sf0005切割TS后片段约254nt。片段切割效率计算公式：切割效率＝切割峰面积/(切割峰面积+未切割峰面积)。
本实施例中选择靶核酸为双链DNA(PCR产物)，引物：
XQ0001-5FAM：GTATGTTGTGTGGAATTGTG 5'6-FAM；
XQ0002-5ROX：GCTGCGCGTAACCACCACAC 5'ROX
扩增产物序列为：GTATGTTGTGTGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGCTATGACCATGATTACGCCAAGCTGCCCTTCATTAGATCTGTGTGGCCAATTC TCCCCTACGTGCTGCTGAAGTTGCAAGGGCAGCTTCAATTCGCCCTATAGTGAGTCGTATTACAATTCACTGGCCGTCGTTTTACAACGTCGTGACTGGGAAAACCCTGGCGTTACCCAACTTAATCGCCTTGCAGCACATCCCCCTTTCGCCAGCTGGCGTAATAGCGAAGAGGCCCGCACCGATCGCCCTTCCCAACAGTTGCGCAGCCTGAATGGCGAATGGACGCGCCCTGTAGCGGCGCATTAAGCGCGGCGGGTGTGGTGGTTACGCGCAGC；斜体部分为PAM序列，下划线区域为靶向区。
Cas-sf0005-5spacer1:GCCGUCAACGUUCAACGCUUGCUCGGUUCGCCGAGAC UCC CCUACGUGCUGCUGAAG(下划线区域为靶向区)
采用如下反应体系：20μL体系，Tris-HAC 40mM、Mg(AC)2 30mM、BSA 120μg/ml、DTT 12mM、pH7.0，Cas-sf0005 50nM，gRNA 100nM，双链靶核酸1μL(PCR产物)。37℃孵育5min、15min、30min、60min，85℃孵育20min。毛细管电泳检测(ABI  3730xl遗传分析仪)FAM、ROX通道中的产物。软件Gene mapper 4.1进行数据分析，计算NTS/TS的切割效率。
结果如图4所示：Cas-sf0005在切割双链靶核酸时，同时切割NTS和TS，切割效率基本一致。
实施例6.Cas-sf0005蛋白的切割特性-切割位置
本实施例通过体外检测测定Cas-sf0005蛋白对双链靶核酸互补链与非互补链的切割位置。本实施例中利用gRNA引导Cas-sf0005蛋白识别并结合在双链靶核酸上；Cas-sf0005蛋白激发对双链靶核酸的切割活性，从而切割体系里的双链靶核酸。切割后的双链靶核酸进行补平加A后连接带T的接头，连接产物进行PCR富集后sanger测序。
本实施例中选择靶核酸为双链DNA(质粒)，合成序列：CATTAGATCTGTGTGGCCAATTC TCCCCTACGTGCTGCTGAAGTTGC连入载体T-Vector-pEASY-Blunt Simple Cloning Vector，斜体部分为PAM序列，下划线区域为靶向区。
gRNA：Cas-sf0005-5spacer1:GCCGUCAACGUUCAACGCUUGCUCGGUUCGCCGAGAC UCCCCUACGUGCUGCUGAAG(下划线区域为靶向区)。
采用如下反应体系：50μL体系，缓冲液：Tris-HAC 40mM、Mg(AC)2 30mM、BSA120μg/ml、DTT 12mM、pH7.0，Cas终浓度为100nM，gRNA终浓度为250nM，双链靶核酸10ng/μL(质粒)。Cas蛋白、gRNA，25℃孵育10min；加入双链靶核酸，37℃孵育1h，85℃孵育5min；上述体系中添加50uL 2X Taq DNA Polymerase Mix(诺唯赞)(1:1)，72℃反应30min；上述反应液进行液体回收；回收液体加入2μL已退火的引物2μM(TK-117：CGGCATTCCTGCTGAACCGCTCTTCCGATCT、TK-111：GATCGGAAGAGC GGTTCAGCAGGAATGCCG)，T4(NEB)连接酶22℃1h。取连接产物10μL，引物S1-PAM-after：ACTCAGCGGCATTCCTGCTGAACCGC、PQ0275-F：CCGTATTACCGCCT TTGAG，2X Taq DNA Polymerase Mix(诺唯赞)进行PCR反应。PCR产物进行sanger测序。结果如图5所示，Cas-sf0005蛋白在切割靶核酸时，切割位置在非互补链NTS的15-16nt中间和互补链TS的21-22nt中间。即，Cas-sf0005对靶序列的互补链的切割位点在PAM互补序列5’端第21nt和22nt之间，Cas-sf0005对靶序列的非互补链的切割位点在PAM序列3’端第15nt和16nt之间，gRNA引导Cas-sf0005蛋白识别并结合上述互补链上，上述非互补链是与互补链配对的DNA链。
实施例7.Cas-sf9417蛋白PAM鉴定
构建Cas-sf9417蛋白表达质粒：对核酸序列进行人源密码子优化后进行基因合成，连入大肠杆菌表达载体PeT28(a)+载体。载体PeT28(a)+-Cas-sf9417中加入JM23119启动子启动Cas-sf9417CrRNA转录。形成载体：PeT28(a)+-Cas-sf9417-JM23119-crRNA，crRNA序列：GUGGGAACCCUUCCUGAUGGCUCGAUCCGUCGAGAC UCCCCUACGUGCUGC UGAAGUUGC下划线为靶点序列；PAM文库的构建：合成序列CGTGTTTCGTAAAGTCTGGAAACGCGGAAGCCCCCAGCGCTTCAGCGTTCNNNNNN TCCCCTACGTGCTGCTG AAGTTGCCCGCAA，N为随机脱氧核苷酸，下划线为靶点序列。经Klenow酶补平后连入pacyc184载体。转化大肠杆菌后提取质粒形成PAM文库。
PAM文库消减实验：制备感受态：BL21(DE3)-PeT28(a)+-Cas-sf9417-JM23119-crRNA。PAM文库质粒转化感受态：BL21(DE3)-PeT28(a)+-Cas-sf9417-JM23119-crRNA，涂布在含有卡那霉素、氯霉素的LB平板上，37℃过夜培养后收集菌体，调整菌液浓度至OD600 0.6-0.8，加IPTG 0.2mM,37℃诱导4h。FastPure EndoFree Plasmid Maxi Kit(vazyme)进行质粒提取，获得消减后PAM库。引物：PAM-F：GGTCTTCGGTTTCCGTGTT；PAM-R：TGGCGTTGACTCTCAGTCAT。以30ng/μL质粒(PAM文库)为模板引物进行PCR反应获得对照组样品，以30ng/μL质粒(消减后PAM库)为模板进行PCR 反应获得实验组样品。对照组样品、实验组样品送二代测序进行数据分析，使用Weblogo作图，Cas-sf9417识别的PAM结构为5’-TTN-3’，其中，N＝A/T/C/G，如图6所示。
实施例8.Cas-sf9417蛋白在进行双链核酸编辑的应用
本实施例体外检测Cas-sf9417的双链DNA的cis切割活性。本实施例中利用可以与靶核酸配对的gRNA引导Cas-sf9417蛋白识别并结合在靶核酸上，从而切割体系里的靶核酸，切割后的靶核酸进行琼脂糖电泳检测。
本实施例中选择靶核酸为双链DNA(质粒)，5spacer1-PAM，其序列为：CATTAGATCTGTGTGGCCAANNN TCCCCTACGTGCTGCTGAAGTTGC连入载体T-Vector-pEASY-Blunt Simple Cloning Vector；斜体部分为PAM序列，N＝A/T/C/G,下划线区域为靶向区。
gRNA:Cas-sf9417-5spacer1:GUGGGAACCCUUCCUGAUGGCUCGAUCCGUCGAGAC UC CCCUACGUGCUGCUGAAG(下划线区域为靶向区)
采用如下反应体系：20μL体系，缓冲液：Tris-HAC 40mM、Mg(AC)2 30mM、BSA 120μg/ml、DTT 12mM、pH7.0，Cas-sf9417终浓度为100nM，gRNA终浓度为200nM，双链靶核酸终浓度为5ng/μL。37℃孵育1h、85℃孵育20min。切割产物进行琼脂糖电泳，检测Cas-sf9417切割能力。实验组添加Cas-sf9417蛋白、gRNA和靶核酸，对照组(CK)不添加gRNA。
结果如图7所示，与不加gRNA的对照相比，PAM为TTN的实验组中Cas-sf9417能够切割体系里的双链核酸，表现出明显的切割条带。这表明Cas-sf9417可以用于PAM为TTN的双链靶核酸的切割和编辑。
实施例9.Cas-sf9417蛋白在动物细胞中的编辑效率
在动物细胞中验证Cas-sf9417蛋白的基因编辑的活性，针对中国仓鼠卵巢细胞(CHO)FUT8基因设计靶点。载体pcDNA3.3经改造后带有EGFP荧光蛋白及PuroR抗性基因。经酶切位点BsmB1插入SV40NLS-Cas-sf9417-NLS融合蛋白；经酶切位点Mfe1插入U6启动子及gRNA序列。CMV启动子启动融合蛋白SV40NLS-Cas-sf9417-NLS-GFP表达。蛋白Cas-sf9417-NLS与蛋白GFP用连接肽T2A进行连接。启动子EF-1α启动嘌呤霉素抗性基因表达。
铺板：CHO细胞融合度至70-80％进行铺板，12孔板中接种细胞数为8*10^4细胞/孔。
转染：铺板12-24h进行转染，100μL opti-MEM中加入2ug质粒混匀；稀释好的质粒加入4μL
Figure PCTCN2022126671-appb-000005
EL Transfection Reagent(TRAN)，室温孵育15-20min。孵育好的混合液加入铺有细胞的培养基中进行转染。
加嘌呤霉素筛选：转染24h加嘌呤霉素，终浓度10ng/ml。嘌呤霉素处理24h更换成正常培养基继续培养24h。
提DNA、PCR扩增编辑区附近、送hiTOM测序：细胞经胰酶消化处理后进行收集，经细胞/组织基因组DNA提取试剂盒(百泰克)进行基因组DNA提取。基因组DNA经引物NAFUT8-JC-Hitom-1F2:GGAGTGAGTACGGTGTGCAGTCCATGTCAGACGCACTG；PQ0106-FUT8-HiTom-R5：GAGTTGGATGCTGGATGGTACAGAACCACTTGTTGGTC扩增靶点附近区域。PCR产物进行hiTOM测序(http://121.40.237.174/HiTOM/Sample_acc eptance_sanyang.php)。
测序数据分析，统计靶点-18位置的上下游20bp范围内的序列种类及比例，得到Cas-sf9417蛋白对靶点位置的编辑效率。
CHO细胞FUT8基因靶点序列：gR16-FUT8：TTT AACAAAGAAGGGTCATCAGTGG CC，斜体部分为PAM序列，下划线区域为靶向区。gRNA序列为GUGGGAACCCUUCCUGAUGGCUCGAUCCGUCGAGAC AACAAAGAAGGGUCAUCAGUGG，下划线区域为靶向 区。
分析结果显示，Cas-sf9417在CHO细胞的靶点gR16-Cas12i3-target-FUT8中的编辑效率为11.39％，编辑类型为InDel，经编辑后的靶核酸的部分测序结果如图8所示。
实施例10.Cas-sf9417蛋白的切割特性-NTS/TS切割效率
本实施例体外检测Cas-sf9417对靶核苷酸双链DNA互补链(TS)、非互补链(NTS)的切割效率。5'6-FAM标记非互补链(NTS)、5'ROX标记互补链(TS)，gRNA引导Cas-sf9417蛋白识别并结合在靶核酸的互补链上，从而切割体系里的靶核酸，切割后的靶核酸进行毛细管电泳检测(ABI 3730xl遗传分析仪)。DNA片段在凝胶中从阴极向阳极迁移，按片段长度大小排列，当迁移到阳极端的激光扫描仪的扫描窗口，荧光染料受到激发，发出一定波长的光，按荧光强度记录下来，每一个带荧光染料的DNA片段电泳轨迹按各自通过激光扫描窗口的实际时间被记录下来，以荧光吸收峰表示每一个片段。峰值越高，表示该片段量越多；峰出现的时间与片段大小有直接关系，片段越小，峰越早出现。FAM荧光通道中Cas-sf9417未切割NTS片段大小为380nt，Cas-sf9417切割NTS后片段约126nt。ROX荧光通道中Cas-sf9417未切割TS片段大小为380nt，Cas-sf9417切割TS后片段约254nt。片段切割效率计算公式：切割效率＝切割峰面积/(切割峰面积+未切割峰面积)。
本实施例中选择靶核酸为双链DNA(PCR产物)，引物：
XQ0001-5FAM：GTATGTTGTGTGGAATTGTG 5'6-FAM；
XQ0002-5ROX：GCTGCGCGTAACCACCACAC 5'ROX
扩增产物序列为：GTATGTTGTGTGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGCTATGACCATGATTACGCCAAGCTGCCCTTCATTAGATCTGTGTGGCCAATTC TCCCCTACGTGCTGCTGAAGTTGCAAGGGCAGCTTCAATTCGCCCTATAGTGAGTCGTATTACAATTCACTGGCCGTCGTTTTACAACGTCGTGACTGGGAAAACCCTGGCGTTACCCAACTTAATCGCCTTGCAGCACATCCCCCTTTCGCCAGCTGGCGTAATAGCGAAGAGGCCCGCACCGATCGCCCTTCCCAACAGTTGCGCAGCCTGAATGGCGAATGGACGCGCCCTGTAGCGGCGCATTAAGCGCGGCGGGTGTGGTGGTTACGCGCAGC；斜体部分为PAM序列，下划线区域为靶向区。
gRNA：Cas-sf9417-5spacer1:GUGGGAACCCUUCCUGAUGGCUCGAUCCGUCGAGAC UCCCCUACGUGCUGCUGAAG(下划线区域为靶向区)
采用如下反应体系：20μL体系，缓冲液：Tris-HAC 40mM、Mg(AC)2 30mM、BSA 120μg/ml、DTT 12mM、pH7.0，Cas-sf9417 50nM，gRNA 100nM，双链靶核酸1μL(PCR产物)。37℃孵育5min、15min、30min、60min，85℃孵育20min。毛细管电泳检测(ABI 3730xl遗传分析仪)FAM、ROX通道中的产物。软件Gene mapper 4.1进行数据分析，计算NTS和TS的切割效率。
结果如图9所示：Cas-sf9417在切割靶核酸时，优先切割NTS。
实施例11.Cas-sf9417蛋白的切割特性-切割位置
本实施例通过体外检测测定Cas-sf9417蛋白对双链靶核酸互补链与非互补链的切割位置。本实施例中利用gRNA引导Cas-sf9417蛋白识别并结合在双链靶核酸上；Cas蛋白激发对双链靶核酸的cis切割活性，从而切割体系里的双链靶核酸。切割后的双链靶核酸进行补平加A后连接带T的接头，连接产物进行PCR富集后sanger测序。
本实施例中选择靶核酸为双链DNA(质粒)，序列：CATTAGATCTGTGTGGCCAATTC TCCCCTACGTGCTGCTGAAGTTGC连入载体T-Vector-pEASY-Blunt Simple Cloning Vector，斜体部分为PAM序列，下划线区域为靶向区。
gRNA：Cas-sf9417-5spacer1:GUGGGAACCCUUCCUGAUGGCUCGAUCCGUCGAGAC UCCCCUACGUGCUGCUGAAG(下划线区域为靶向区)。
采用如下反应体系：50μL体系，缓冲液：Tris-HAC 40mM、Mg(AC)2 30mM、BSA 120μg/ml、DTT 12mM、pH7.0，Cas-sf9417 100nM，gRNA 250nM，双链靶核酸10ng/μL(质粒)。Cas蛋白、gRNA，25℃孵育10min；加入双链靶核酸，37℃孵育1h，85℃孵育5min；上述体系中添加50uL 2X Taq DNA Polymerase Mix(诺唯赞)(1:1)，72℃反应30min；上述反应液进行液体回收；回收液体加入2μL已退火的引物2μM(TK-117：CGGCATTCCTGCTGAACCGCTCTTCCGATCT、TK-111：GATCGGAAGAGCGGTTCAGCAGGAATGCCG)，T4(NEB)连接酶22℃1h。取连接产物10μL，引物S1-PAM-after：ACTCAGCGGCATTCCTGCTGAACCGC、PQ0275-F：CCGTATTACCGCCTTTGAG，2X Taq DNA Polymerase Mix(诺唯赞)进行PCR反应。PCR产物进行sanger测序。结果如图10所示，Cas-sf9417蛋白在切割靶核酸时，切割位置在NTS的14-16nt中间和TS的21-22nt中间。即，Cas-sf9417对靶序列的互补链的切割位点在PAM互补序列5’端第21nt和22nt之间，Cas-sf9417对靶序列的非互补链的切割位点在PAM序列3’端第14nt和16nt之间，gRNA引导Cas-sf9417蛋白识别并结合上述互补链上，上述非互补链是与互补链配对的DNA链。
实施例12.Cas-sf8553蛋白PAM鉴定
构建Cas-sf8553蛋白表达质粒：对核酸序列进行人源密码子优化后进行基因合成，连入大肠杆菌表达载体PeT28(a)+载体。载体PeT28(a)+-Cas-sf8553中加入JM23119启动子启动Cas-sf8553CrRNA转录。形成载体：PeT28(a)+-Cas-sf8553-JM23119-crRNA，crRNA序列：GUGAGGACGACAACCGAAGGCUCGGUGCGCCGAGAC UCCCCUACGUGCUGC UGAAGUUGC下划线为靶点序列；PAM文库的构建：合成序列CGTGTTTCGTAAAGTCTGGAAACGCGGAAGCCCCCAGCGCTTCAGCGTTCNNNNNN TCCCCTACGTGCTGCTG AAGTTGCCCGCAA，N为随机脱氧核苷酸，下划线为靶点序列。经Klenow酶补平后连入pacyc184载体。转化大肠杆菌后提取质粒形成PAM文库。
PAM文库消减实验：制备感受态：BL21(DE3)-PeT28(a)+-Cas-sf8553-JM23119-crRNA。PAM文库质粒转化感受态：BL21(DE3)-PeT28(a)+-Cas-sf8553-JM23119-crRNA，涂布在含有卡那霉素、氯霉素的LB平板上，37℃过夜培养后收集菌体，调整菌液浓度至OD600 0.6-0.8,加IPTG 0.2mM,37℃诱导4h。大提试剂盒进行质粒提取，获得消减后PAM库。引物：PAM-F：GGTCTTCGGTTTCCGTGTT；PAM-R：TGGCGTTGACTCTCAGTCAT。以30ng/μL质粒(PAM文库)为模板，进行PCR反应获得对照组样品，以30ng/μL质粒(消减后PAM库)为模板进行PCR反应获得实验组样品。对照组样品、实验组样品送二代测序进行数据分析，使用Weblogo作图，Cas-sf8553识别的PAM结构为5’-TTN-3’，N＝A/T/C/G，如图11所示。
实施例13.Cas-sf8553蛋白在进行双链核酸编辑的应用
本实施例检测Cas-sf8553的双链DNA的cis切割活性。本实施例中利用可以与靶核酸配对的gRNA引导Cas-sf8553蛋白识别并结合在靶核酸上，从而切割体系里的靶核酸，切割后的靶核酸进行琼脂糖电泳检测。
本实施例中选择靶核酸为双链DNA(质粒)，5spacer1-PAM，其序列为：CATTAGATCTGTGTGGCCAANNN TCCCCTACGTGCTGCTGAAGTTGC连入载体T-Vector-pEASY-Blunt Simple Cloning Vector；斜体部分为PAM序列，N＝A/T/C/G,下划线区域为靶向区。
Cas-sf8553-5spacer1:GUGAGGACGACAACCGAAGGCUCGGUGCGCCGAGAC UCCC CUACGUGCUGCUGAAG(下划线区域为靶向区)
采用如下反应体系：20μL体系，缓冲液：Tris-HAC 40mM、Mg(AC)2 30mM、BSA 120μg/ml、DTT 12mM、pH7.0，Cas-sf8553 100nM，gRNA 200nM，双链靶核酸终浓度为5ng/μL。37℃孵育1h、85℃孵育20min。切割产物用1％琼脂糖进行电泳，检测Cas-sf8553切割能力。实验组添加Cas-sf8553蛋白、gRNA和靶核酸，对照组(CK)不添加gRNA。
结果如图12所示，与不加gRNA的对照相比，PAM为TTN的实验组中Cas-sf8553能够切割体系里的双链核酸，表现出明显的切割条带。这表明Cas-sf8553可以用于PAM为TTN的双链靶核酸的切割和编辑。
实施例14.Cas-sf8553蛋白在动物细胞中的编辑效率
采用Cas-sf8553蛋白在动物细胞中验证其基因编辑的活性，针对中国仓鼠卵巢细胞(CHO)FUT8基因设计靶点。载体pcDNA3.3经改造后带有EGFP荧光蛋白及PuroR抗性基因。经酶切位点BsmB1插入SV40NLS-Cas-sf8553-NLS融合蛋白；经酶切位点Mfe1插入U6启动子及gRNA序列。CMV启动子启动融合蛋白SV40NLS-Cas-sf8553-NLS-GFP表达。蛋白Cas-sf8553-NLS与蛋白GFP用连接肽T2A进行连接。启动子EF-1α启动嘌呤霉素抗性基因表达。
铺板：CHO细胞融合度至70-80％进行铺板，12孔板中接种细胞数为8*10^4细胞/孔。
转染：铺板12-24h进行转染，100μL opti-MEM中加入2ug质粒混匀；稀释好的质粒加入4μL
Figure PCTCN2022126671-appb-000006
EL Transfection Reagent(TRAN)，室温孵育15-20min。孵育好的混合液加入铺有细胞的培养基中进行转染。
加嘌呤霉素筛选：转染24h加嘌呤霉素，终浓度10ng/ml。嘌呤霉素处理24h更换成正常培养基继续培养24h。
提DNA、PCR扩增编辑区附近、送hiTOM测序：细胞经胰酶消化处理后进行收集，经细胞/组织基因组DNA提取试剂盒(百泰克)进行基因组DNA提取。基因组DNA经引物PQ0106-FUT8-HiTom-F1：GGAGTGAGTACGGTGTGCGAGTTCTGTTGCATGGTAGG；PQ0106-FUT8-HiTom-R1：GAGTTGGATGCTGGATGGGCCAAGCTTCTTGGTGGTTTC扩增靶点附近区域。PCR产物进行hiTOM测序(http://121.40.237.174/HiTOM/Sample_acceptance_sanyang.php)。
测序数据分析，统计靶点-18位置的上下游20bp范围内的序列种类及比例，得到Cas-sf0005蛋白对靶点位置的编辑效率。
CHO细胞FUT8基因靶点序列：gR3-FUT8：TTC CAGCCAAGGTTGTGGACGGATC A，斜体部分为PAM序列，下划线区域为靶向区。gRNA序列为GUGAGGACGACAACCGAAGGCUCGGUGCGCCGAGAC CAGCCAAGGUUGUGGACGGAUCA，下划线区域为靶向区。
分析结果显示，Cas-sf8553在CHO细胞的靶点gR3-FUT8中的编辑效率为1.05％，编辑类型为InDel。
实施例15.Cas-sf8553蛋白的切割特性-NTS/TS切割效率
本实施例检测Cas-sf8553对靶核苷酸(双链DNA)互补链(TS)、非互补链(NTS)的切割效率。5'6-FAM标记非互补链(NTS)、5'ROX标记互补链(TS)，gRNA引导Cas-sf8553蛋白识别并结合在靶核酸上，从而切割体系里的靶核酸，切割后的靶核酸进行毛细管电泳检测(ABI 3730xl遗传分析仪)。DNA片段在凝胶中从阴极向阳极迁移，按片段长度大小排列，当迁移到阳极端的激光扫描仪的扫描窗口，荧光染料受到激发，发出一定波长的光，按荧光强度记录下来，每一个带荧光染料的DNA片段电泳轨迹按各自通过激光扫描窗口的实际时间被记录下来，以荧光吸收峰表示每一个片段。峰值越高，表 示该片段量越多；峰出现的时间与片段大小有直接关系，片段越小，峰越早出现。FAM荧光通道中Cas-sf8553未切割NTS片段大小为380nt，Cas-sf8553切割NTS后片段约126nt。ROX荧光通道中Cas-sf8553未切割TS片段大小为380nt，Cas-sf8553切割TS后片段约254nt。片段切割效率计算公式：切割效率＝切割峰面积/(切割峰面积+未切割峰面积)
本实施例中选择靶核酸为双链DNA(PCR产物)，引物：
XQ0001-5FAM：GTATGTTGTGTGGAATTGTG 5'6-FAM；
XQ0002-5ROX：GCTGCGCGTAACCACCACAC 5'ROX
扩增产物序列为：GTATGTTGTGTGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGCTATGACCATGATTACGCCAAGCTGCCCTTCATTAGATCTGTGTGGCCAATTC TCCCCTACGTGCTGCTGAAGTTGCAAGGGCAGCTTCAATTCGCCCTATAGTGAGTCGTATTACAATTCACTGGCCGTCGTTTTACAACGTCGTGACTGGGAAAACCCTGGCGTTACCCAACTTAATCGCCTTGCAGCACATCCCCCTTTCGCCAGCTGGCGTAATAGCGAAGAGGCCCGCACCGATCGCCCTTCCCAACAGTTGCGCAGCCTGAATGGCGAATGGACGCGCCCTGTAGCGGCGCATTAAGCGCGGCGGGTGTGGTGGTTACGCGCAGC；斜体部分为PAM序列，下划线区域为靶向区。
gRNA：Cas-sf8553-5spacer1:GUGAGGACGACAACCGAAGGCUCGGUGCGCCGAGAC UCCCCUACGUGCUGCUGAAG(下划线区域为靶向区)。
采用如下反应体系：20μL体系，缓冲液：Tris-HAC 40mM、Mg(AC)2 30mM、BSA 120μg/ml、DTT 12mM、pH7.0，Cas-sf8553 50nM，gRNA 100nM，双链靶核酸1μL(PCR产物)。37℃孵育5min、15min、30min、60min，85℃孵育20min。毛细管电泳检测(ABI 3730xl遗传分析仪)FAM、ROX通道中的产物。软件Gene mapper 4.1进行数据分析，计算NTS/TS的切割效率。
结果如图13所示：Cas-sf8553在切割靶核酸时，同时切割NTS和TS，切割效率基本一致。
实施例16.Cas-sf8553蛋白的切割特性-切割位置
本实施例通过体外检测测定Cas-sf8553蛋白双链靶核酸互补链与非互补链的切割位置。本实施例中利用gRNA引导Cas-sf8553蛋白识别并结合在双链靶核酸上；Cas-sf8553蛋白激发对双链靶核酸的cis切割活性，从而切割体系里的双链靶核酸。切割后的双链靶核酸进行补平加A后连接带T的接头，连接产物进行PCR富集后sanger测序。
本实施例中选择靶核酸为双链DNA(质粒)，序列：CATTAGATCTGTGTGGCCAATTC TCCCCTACGTGCTGCTGAAGTTGC连入载体T-Vector-pEASY-Blunt Simple Cloning Vector，斜体部分为PAM序列，下划线区域为靶向区。
gRNA：Cas-sf8553-5spacer1:GUGAGGACGACAACCGAAGGCUCGGUGCGCCGAGAC UCCCCUACGUGCUGCUGAAG(下划线区域为靶向区)
采用如下反应体系：50μL体系，缓冲液：Tris-HAC 40mM、Mg(AC)2 30mM、BSA 120μg/ml、DTT 12mM、pH7.0，Ca-sf8553 100nM，gRNA 250nM，双链靶核酸10ng/μL(质粒)。Cas蛋白、gRNA，25℃孵育10min；加入双链靶核酸，37℃孵育1h，85℃孵育5min；上述体系中添加50uL 2X Taq DNA Polymerase Mix(诺唯赞)(1:1)，72℃反应30min；上述反应液进行液体回收；回收液体加入2μL已退火的引物2μM(TK-117：CGGCATTCCTGCTGAACCGCTCTTCCGATCT、TK-111：GATCGGAAGAGCGGTTCAGCAGGAATGCCG)，T4(NEB)连接酶22℃1h。取连接产物10μL，引物S1-PAM-after：ACTCAGCGGCATTCCTGCTGAACCGC、PQ0275-F：CCGTATTACCGCCTTTGAG，2X Taq DNA Polymerase Mix(诺唯赞)进行PCR反应。PCR产物进行sanger 测序。结果如图14所示，Cas-sf8553蛋白切割靶核酸时，切割的位置为NTS 14-15nt中间和TS 21-23nt中间。即，Cas-sf8553对靶序列的互补链的切割位点在PAM互补序列5’端第21nt和23nt之间，Cas-sf8553对靶序列的非互补链的切割位点在PAM序列3’端第14nt和15nt之间，gRNA引导Cas-sf8553蛋白识别并结合上述互补链上，上述非互补链是与互补链配对的DNA链。
实施例17.Cas-sf1846蛋白在进行核酸检测时的应用
本实施例通过体外检测试验以验证Cas-sf1846的trans切割活性。本实施例中利用可以与靶核酸配对的gRNA引导Cas-sf1846蛋白识别并结合在靶核酸上；随后，Cas-sf1846蛋白激发对任意单链核酸的trans切割活性，从而切割体系里的单链核酸检测器；单链核酸检测器的两端分别设置荧光基团和淬灭基团，如果单链核酸检测器被切割，则会激发荧光；在其他的实施方式中，单链核酸检测器的两端还可以设置成能够被胶体金检测的标记。
本实施例中选择靶核酸为单链DNA，N-B-i3g1-ssDNA0，其序列为：CGACATTCCGAAGAACGCTGAAGCGCTGGGGGCAAATTGTGCAATTTGCGGC；
Cas-sf1846所对应的gRNA序列为:GUUGCAAGCCUUACUGAUUGCUCCUCACGAGGAGAC CCCCCAGCGCUUCAGCGUUC(下划线区域为gRNA的靶向区)；
单链核酸检测器序列为FAM-TTATT-BHQ1；
采用如下反应体系：Cas-sf1846终浓度为50nM，gRNA终浓度为50nM，靶核酸终浓度为50nM,单链核酸检测器终浓度200nM。37℃孵育，读取FAM荧光/1min。对照组不添加靶核酸。
如图15所示，与不加靶核酸的对照相比，在有靶核酸存在的情况下，Cas-sf1846切割体系里的单链核酸检测，快速的报告出荧光。以上实验反映出，配合单链核酸检测器，Cas-sf1846可以用于靶核酸的检测。图15中，1为添加靶核酸的实验结果，2为不添加靶核酸的对照组。
尽管本发明的具体实施方式已经得到详细的描述，但本领域技术人员将理解：根据已经公布的所有教导，可以对细节进行各种修改和变动，并且这些改变均在本发明的保护范围之内。本发明的全部分为由所附权利要求及其任何等同物给出。

Claims (22)

  1. 一种Cas蛋白，其特征在于，所述Cas蛋白为以下I-III任一所述的Cas蛋白：
    I、Cas蛋白的氨基酸序列与SEQ ID No.1或SEQ ID No.4或SEQ ID No.7或SEQ ID No.10相比，具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、或至少99％的序列同一性，并且基本保留了其源自的序列的生物学功能；
    II、所述Cas蛋白的氨基酸序列与SEQ ID No.1或SEQ ID No.4或SEQ ID No.7或SEQ ID No.10相比，具有一个或多个氨基酸的置换、缺失或添加的序列，并且基本保留了其源自的序列的生物学功能；
    III、所述Cas蛋白包含SEQ ID No.1或SEQ ID No.4或SEQ ID No.7或SEQ ID No.10所示的氨基酸序列。
  2. 一种融合蛋白，所述融合蛋白包括权利要求1所述的Cas蛋白和其他的修饰部分。
  3. 一种分离的多核苷酸，其特征在于，所述多核苷酸为编码权利要求1所述Cas蛋白的多核苷酸序列，或编码权利要求2所述融合蛋白的多核苷酸序列。
  4. 一种gRNA，其特征在于，所述gRNA包括能够结合权利要求1所述的Cas蛋白的同向重复序列和能够靶向靶序列的引导序列。
  5. 一种同向重复序列，其特征在于，所述同向重复序列包含SEQ ID No.13-16任一所示的序列。
  6. 一种载体，其特征在于，所述载体包含权利要求3所述的多核苷酸以及与之可操作连接的调控元件。
  7. 一种CRISPR-Cas系统，其特征在于，所述系统包括权利要求1所述的Cas蛋白以及至少一种权利要求4所述的gRNA。
  8. 一种载体系统，其特征在于，所述载体系统包括一种或多种载体，该一种或多种载体包括：
    a)第一调控元件，该第一调控元件可操作地与权利要求4所述的gRNA连接，
    b)第二调控元件，该第二调控元件可操作地与权利要求1所述的Cas蛋白连接；
    其中组分(a)和(b)位于该系统的相同或不同载体上。
  9. 一种组合物，其特征在于，所述组合物包含：
    (i)蛋白组分，其选自：权利要求1所述的Cas蛋白或权利要求2所述的融合蛋白；
    (ii)核酸组分，其选自：权利要求4所述的gRNA，或编码权利要求4所述的gRNA的核酸，或权利要求4所述的gRNA的前体RNA，或编码权利要求4所述的gRNA的前体RNA核酸；
    所述蛋白组分与核酸组分相互结合形成复合物。
  10. 一种活化的CRISPR复合物，所述活化的CRISPR复合物包含：
    (i)蛋白组分，其选自：权利要求1所述的Cas蛋白或权利要求2所述的融合蛋白；
    (ii)核酸组分，其选自：权利要求4所述的gRNA，或编码权利要求4所述的gRNA的核酸，或权利要求4所述的gRNA的前体RNA，或编码权利要求4所述的gRNA的前体RNA核酸；
    (iii)结合在权利要求4所述的gRNA上的靶序列。
  11. 一种工程化的宿主细胞，其特征在于，所述宿主细胞包含权利要求1所述的Cas蛋白，或权利要求2所述的融合蛋白，或权利要求3所述的多核苷酸，或权利要求6所述的载体，或权利要求7所述的CRISPR-Cas系统，或权利要求8所述的载体系统，或权利 要求9所述的组合物，或权利要求10所述的活化的CRISPR复合物。
  12. 权利要求1所述的Cas蛋白，或权利要求2所述的融合蛋白，或权利要求3所述的多核苷酸，或权利要求6所述的载体，或权利要求7所述的CRISPR-Cas系统，或权利要求8所述的载体系统，或权利要求9所述的组合物，或权利要求10所述的活化的CRISPR复合物，或权利要求11所述的宿主细胞在基因编辑、基因靶向或基因切割中的应用；或者，在制备用于基因编辑、基因靶向或基因切割的试剂或试剂盒中的应用。
  13. 根据权利要求12所述的应用，所述应用为基因编辑或基因切割中的应用，或者，在制备用于基因编辑或基因切割的试剂或试剂盒中的应用；其特征在于，
    所述基因包含互补链和非互补链，所述gRNA引导Cas蛋白识别并结合在所述互补链上，所述非互补链是与互补链配对的核酸链，所述非互补链上含有PAM序列，所述互补链含有与上述PAM序列配对的PAM互补序列；
    所述Cas蛋白在靶序列的互补链的PAM互补序列5’端第21nt和22nt之间进行切割，或者，所述Cas蛋白在靶序列的非互补链的PAM序列3’端第15nt和16nt之间进行切割；
    或者，所述Cas蛋白在靶序列的互补链的PAM互补序列5’端第21nt和22nt之间进行切割，或者，所述Cas蛋白在靶序列的非互补链的PAM序列3’端第14nt和16nt之间进行切割；
    或者，所述Cas蛋白在靶序列的互补链的PAM互补序列5’端第21nt和23nt之间进行切割，或者，所述Cas蛋白在靶序列的非互补链的PAM序列3’端第14nt和15nt之间进行切割。
  14. 权利要求1所述的Cas蛋白，或权利要求2所述的融合蛋白，或权利要求3所述的多核苷酸，或权利要求6所述的载体，或权利要求7所述的CRISPR-Cas系统，或权利要求8所述的载体系统，或权利要求9所述的组合物，或权利要求10所述的活化的CRISPR复合物，或权利要求11所述的宿主细胞在选自如下任一或任意几种中的应用：
    靶向和/或编辑靶核酸；切割双链DNA、单链DNA或单链RNA；非特异性切割和/或降解侧枝核酸；非特异性的切割单链核酸；核酸检测；特异性地编辑双链核酸；碱基编辑双链核酸；碱基编辑单链核酸。
  15. 根据权利要求14所述的应用，其特征在于，所述应用为切割双链DNA，
    所述双链DNA包含互补链和非互补链，所述gRNA引导Cas蛋白识别并结合在所述互补链上，所述非互补链是与互补链配对的核酸链，所述非互补链上含有PAM序列，所述互补链含有与上述PAM序列配对的PAM互补序列；
    所述Cas蛋白在靶序列的互补链的PAM互补序列5’端第21nt和22nt之间进行切割，或者，所述Cas蛋白在靶序列的非互补链的PAM序列3’端第15nt和16nt之间进行切割；
    或者，所述Cas蛋白在靶序列的互补链的PAM互补序列5’端第21nt和22nt之间进行切割，或者，所述Cas蛋白在靶序列的非互补链的PAM序列3’端第14nt和16nt之间进行切割；
    或者，所述Cas蛋白在靶序列的互补链的PAM互补序列5’端第21nt和23nt之间进行切割，或者，所述Cas蛋白在靶序列的非互补链的PAM序列3’端第14nt和15nt之间进行切割。
  16. 一种编辑靶核酸、靶向靶核酸或切割靶核酸的方法，所述方法包括将靶核酸与权利要求1所述的Cas蛋白，或权利要求2所述的融合蛋白，或权利要求3所述的多核苷酸，或权利要求6所述的载体，或权利要求7所述的CRISPR-Cas系统，或权利要求8所述的载体系统，或权利要求9所述的组合物，或权利要求10所述的活化的CRISPR复合 物，或权利要求11所述的宿主细胞进行接触。
  17. 根据权利要求16所述的方法，其特征在于，所述方法为切割靶核酸的方法，
    所述核酸包含互补链和非互补链，所述gRNA引导Cas蛋白识别并结合在所述互补链上，所述非互补链是与互补链配对的核酸链，所述非互补链上含有PAM序列，所述互补链含有与上述PAM序列配对的PAM互补序列；
    所述Cas蛋白在靶序列的互补链的PAM互补序列5’端第21nt和22nt之间进行切割，或者，所述Cas蛋白在靶序列的非互补链的PAM序列3’端第15nt和16nt之间进行切割；
    或者，所述Cas蛋白在靶序列的互补链的PAM互补序列5’端第21nt和22nt之间进行切割，或者，所述Cas蛋白在靶序列的非互补链的PAM序列3’端第14nt和16nt之间进行切割；
    或者，所述Cas蛋白在靶序列的互补链的PAM互补序列5’端第21nt和23nt之间进行切割，或者，所述Cas蛋白在靶序列的非互补链的PAM序列3’端第14nt和15nt之间进行切割。
  18. 一种切割单链核酸的方法，所述方法包括，使核酸群体与权利要求1所述的Cas蛋白和权利要求4所述的gRNA接触，其中所述核酸群体包含靶核酸和至少一个非靶单链核酸，所述gRNA能够靶向所述靶核酸，所述Cas蛋白切割所述非靶单链核酸。
  19. 一种用于基因编辑、基因靶向或基因切割的试剂盒，所述试剂盒包括权利要求1所述的Cas蛋白，或权利要求2所述的融合蛋白，或权利要求3所述的多核苷酸，或权利要求6所述的载体，或权利要求7所述的CRISPR-Cas系统，或权利要求8所述的载体系统，或权利要求9所述的组合物，或权利要求10所述的活化的CRISPR复合物，或权利要求11所述的宿主细胞。
  20. 一种用于检测样品中的靶核酸的试剂盒，所述试剂盒包含：(a)权利要求1所述的Cas蛋白，或编码所述Cas蛋白的核酸；(b)权利要求4所述的gRNA，或编码所述gRNA的核酸，或包含所述gRNA的前体RNA，或编码所述前体RNA的核酸；和(c)为单链的且不与所述gRNA杂交的单链核酸检测器。
  21. 权利要求1所述的Cas蛋白，或权利要求2所述的融合蛋白，或权利要求3所述的多核苷酸，或权利要求6所述的载体，或权利要求7所述的CRISPR-Cas系统，或权利要求8所述的载体系统，或权利要求9所述的组合物，或权利要求10所述的活化的CRISPR复合物，或权利要求11所述的宿主细胞在制备制剂或试剂盒中的用途，所述制剂或试剂盒用于：
    (i)基因或基因组编辑；
    (ii)靶核酸检测和/或诊断；
    (iii)编辑靶基因座中的靶序列来修饰生物或非人类生物；
    (iv)疾病的治疗；
    (v)靶向靶基因；
    (vi)切割目的基因。
  22. 一种检测样品中靶核酸的方法，所述方法包括将样品与权利要求1所述的Cas蛋白、gRNA(指导RNA)和单链核酸检测器接触，所述gRNA包括与所述Cas蛋白结合的区域和与靶核酸杂交的指导序列；检测由所述Cas蛋白切割单链核酸检测器产生的可检测信号，从而检测靶核酸；所述单链核酸检测器不与所述gRNA杂交。
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